OP-3-induced morphogenesis

ABSTRACT

Disclosed are (1) nucleic acid and amino acid sequences for a novel morphogenic protein; (2) methods for producing and expressing the protein in a biologically active form; and (3) methods for utilizing the protein to induce tissue morphogenesis in a mammal, including methods for increasing a progenitor cell population in a mammal, methods for stimulating progenitor cells to differentiate and maintain their differentiated phenotype in vivo or in vitro, methods for inducing tissue-specific growth in vivo and methods for the replacement of diseased or damaged tissue in vivo.

REFERENCE TO RELATED APPLICATIONS

This is a divisional of application Ser. No. 07/971,091 filed on Nov. 3,1992 now abandoned which is a continuation-in-part of the U.S.application Ser. Nos.: 1) U.S. Ser. No. 07/922,813, abandoned, filedJul. 31, 1992, which is a continuation-in-part of U.S. Ser. No.07/752,764, abandoned, filed Aug. 31, 1991, which is acontinuation-in-part of U.S. Ser. No. 07/667,274, abandoned, filed Mar.11, 1991; 2) U.S. Ser. No. 07/923,780, abandoned, filed Jul. 31, 1992,which is a continuation-in-part of U.S. Ser. No. 07/752,764, abandoned,and U.S. Ser. No. 07/752,857, abandoned, both filed Aug. 30, 1991, andboth continuations-in-part of 07/667,274, abandoned; 3) U.S. Ser. No.938,336, abandoned, and U.S. Ser. No. 938,337, abandoned, both filedAug. 28, 1992, and both continuations-in-part of U.S. Ser. No.07/753,059, abandoned, filed Aug. 30, 1991, which is acontinuation-in-part of 07/667,274, abandoned; (4) U.S. Ser. No.07/938,021, abandoned, filed Aug. 28, 1992, which is acontinuation-in-part of U.S. Ser. No. 752,861, abandoned, filed Aug. 30,1991, which is a continuation-in-part of U.S. Ser. No. 667,274,abandoned; (5) U.S. Ser. No. 945,285, abandoned, and U.S. Ser. No.945,286, abandoned, both filed on Sep. 15, 1992 and bothcontinuations-in-part of U.S. Ser. No. 752,764 abandoned; (6) U.S. Ser.No. 946,235, abandoned, and U.S. Ser. No. 946,238, abandoned, both filedSep. 16, 1992 and both continuations-in-part of U.S. Ser. No. 752,764,abandoned. The disclosures of these applications are incorporated byreference herein.

FIELD OF THE INVENTION

This invention relates generally to the field of tissue morphogenesisand more particularly to a novel protein that induces tissuemorphogenesis in mammals.

BACKGROUND OF THE INVENTION

Cell differentiation is the central characteristic of morphogenesiswhich initiates in the embryo, and continues to various degreesthroughout the life of an organism in adult tissue repair andregeneration mechanisms. The degree of morphogenesis in adult tissuevaries among different tissues and is related, among other things, tothe degree of cell turnover in a given tissue. On this basis, tissuescan be divided into three broad categories: (1) tissues with static cellpopulations such as nerve and skeletal muscle where there is no celldivision and most of the cells formed during early development persistthroughout adult life; (2) tissues containing conditionally renewingpopulations such as liver where there is generally little cell divisionbut, in response to an appropriate stimulus, cells can divide to producedaughters of the same differentially defined type; and (3) tissues withpermanently renewing populations including blood, testes and stratifiedsquamous epithelia which are characterized by rapid and continuous cellturnover in the adult. Here, the terminally differentiated cells have arelatively short life span and are replaced through proliferation of adistinct subpopulation of cells, known as stem or progenitor cells.

The cellular and molecular events which govern the stimulus fordifferentiation of these cells is an area of intensive research. In themedical field, it is anticipated that the discovery of factor(s) whichcontrol cell differentiation and tissue morphogenesis will advancesignificantly medicine's ability to repair and regenerate diseased ordamaged mammalian tissues and organs. Particularly useful areas includereconstructive surgery and in the treatment of tissue degenerativediseases including arthritis, emphysema, osteoporosis, cardiomyopathy,cirrhosis, and degenerative nerve diseases.

A number of different factors have been isolated in recent years whichappear to play a role in cell differentiation. Recently, various membersof the structurally related proteins of the transforming growth factor(TGF)-β superfamily of proteins have been identified as true morphogens.

This "family" of proteins, sharing substantial amino acid sequencehomology within their morphogenically active C-terminal domains,including a conserved six or seven cysteine skeleton, are capable ofinducing tissue-specific morphogenesis in a variety of organs andtissues, including bone, cartilage, liver, dentin, periodontal ligament,cementum, nerve tissue and the epithelial mucosa of the gastrointestinaltract. The proteins apparently bind to surface receptors or otherwisecontact and interact with progenitor cells, predisposing or stimulatingthe cells to proliferate and differentiate in a morphogenicallypermissive environment. The morphogens are capable of inducing thedevelopmental cascade of cellular and molecular events that culminate inthe formation of new organ-specific tissue, including anyvascularization, connective tissue formation, and nerve ennervation asrequired by the naturally occurring tissue.

Among the proteins useful in tissue morphogenesis are proteinsoriginally identified as bone inductive proteins, such as the OP-1,(also referred to in related applications as "OP1"), OP-2 (also referredto in related applications as "OP2"), and the CBMP2 proteins, as well asamino acid sequence-related proteins such as BMP5, BMP6 and its murinehomolog, Vgr-1, DPP and 60A (from Drosophila), Vgl (from Xenopus), andGDF-1 (from mouse see, for example, U.S. Ser. No. 752,764, abandoned,U.S. Ser. No. 667,274, abandoned, and U.S. Ser. No. 923,780, abandoned,and PCT documents U.S. Ser. No. 92/01968 and U.S. Ser. No. 92/07358).These TGF-β superfamily members comprise a distinct subfamily ofproteins different from other members of the TGF-β superfamily in thatthe family of morphogenic proteins are able to induce the full cascadeof events that result in tissue morphogenesis, including stimulatingcell proliferation and cell differentiation, supporting the growth andmaintenance of differentiated cells and inducing the "redifferentiation"of transformed cells to display a morphology characteristic ofuntransformed cells. The morphogenic proteins apparently can act asendocrine, paracrine or autocrine factors. Specifically, the endogenousmorphogens may be synthesized by the cells on which they act, byneighboring cells, or by cells of a distant tissue, the secreted proteinbeing transported to the cells to be acted on. In addition, the familyof morphogenic proteins induce true tissue morphogenesis, rather thaninducing formation of fibrotic (scar) tissue as, for example, TGF-βdoes.

The morphogens are synthesized in the cell as a precursor moleculeapproximately three times larger than the mature protein that isprocessed to yield mature disulfide-linked dimers comprising theC-terminal domain of the precursor sequence. The proteins are inactivewhen reduced e.g., in monomeric form, but are active as oxidizedhomodimeric species as well as when oxidized in combination with othermorphogens to produce heterodimers. The proteins useful in tissuemorphogenesis typically require a suitable environment enabling cells tomigrate, proliferate and differentiate in a tissue-specific manner into,e.g., cartilage-producing chondroblasts, bone-producing osteoblasts,hemopoietic cells, or liver cells, depending on the nature of the localenvironment. The proliferation and differentiation of cells induced bythe morphogenic proteins requires a suitable local environment,including a suitable substratum on which the cells can anchor. Theproliferating and differentiating cells also require the presence ofappropriate signals to direct their tissue-specificity, such as cellsurface markers.

It is an object of this invention to provide a novel purifiedmorphogenic protein, "OP-3", including the amino acid sequence definingit and nucleic acids encoding it, including allelic, species, mutant andchimeric variants thereof, and methods for utilizing the protein toinduce the developmental cascade of tissue morphogenesis for a varietyof tissues in mammals. The morphogenic properties of OP-3 include theability to induce proliferation and differentiation of progenitor cells,and the ability to support and maintain the differentiated phenotypethrough the progression of events that results in the formation of adulttissue. Another object is to provide methods for the expression andisolation of morphogenically active species of OP-3 using recombinantDNA techniques. Yet another object is to provide generic sequencesdefining useful morphogens. Still another object is to providetissue-specific acellular matrices that may be used in combination withOP-3, and methods for their preparation. Other objects include utilizingOP-3 in a variety of applications including methods for increasing aprogenitor cell population in a mammal; methods for stimulatingprogenitor cells to differentiate in vivo or in vitro and to maintaintheir differentiated phenotype; methods for inducing tissue-specificgrowth in vivo, and methods for the replacement of diseased or damagedtissue in vivo. These and other objects and features of the inventionwill be apparent from the description, drawings, and claims whichfollow.

SUMMARY OF THE INVENTION

A novel substantially pure genetic sequence encoding a novelsubstantially pure protein referred to herein as "OP-3" now has beendiscovered. This novel protein is a member of the morphogenic proteinfamily previously described by Applicants (see U.S. Ser. No. 667,274,abandoned, and U.S. Ser. No. 752,764, abandoned). Accordingly, theinvention provides methods for utilizing OP-3 to induce thedevelopmental cascade of tissue morphogenesis in a mammal. Specifically,methods are provided for utilizing OP-3 to induce the proliferation ofuncommitted progenitor cells, to induce the differentiation of thesestimulated progenitor cells in a tissue-specific manner underappropriate environmental conditions, and to support the growth andmaintenance of these differentiated cells. The protein also may be usedto stimulate the "redifferentiation" of cells that have strayed fromtheir differentiated phenotypes. Accordingly, OP-3 can be utilized toinitiate and maintain the developmental cascade of tissue morphogenesisin an appropriate, morphogenically permissive environment.

As used herein, useful OP-3 morphogens include proteins encoded by theDNA sequence provided in Seq. ID No. 1 ("mOP-3") and allelic and speciesvariants thereof, as well as other naturally-occurring and biosyntheticmutants, including chimeric proteins, that are morphogenically active asdefined herein. "Morphogenically active fragment" is understood toinclude all proteins and protein fragments encoded by part or all of thesequence of Seq. ID No. 1 and which have morphogenic activity as definedherein. Specifically, as defined herein, a morphogen is a dimericprotein comprising a pair of polypeptide chains, wherein eachpolypeptide chain comprises at least the C-terminal six cysteineskeleton defined by residues 303 to 399 of Seq. ID No. 1 (or residues335-431 of OP1, Seq. ID no. 3), including functionally equivalentarrangements of these cysteines (e.g., amino acid insertions ordeletions which alter the linear arrangement of the cysteines in thesequence but not their relationship in the folded structure), such that,when the polypeptide chains are folded, the dimeric protein speciescomprising the pair of polypeptide chains has the appropriatethree-dimensional structure, including the appropriate intra- orinter-chain disulfide bonds such that the protein is capable of actingas a morphogen as defined herein. Specifically, the morphogens generallyare capable of all of the following biological functions in amorphogenically permissive environment: stimulating proliferation ofprogenitor cells; stimulating the differentiation of progenitor cells;stimulating the proliferation of differentiated cells; and supportingthe growth and maintenance of differentiated cells, including the"redifferentiation" of transformed cells. In addition, it is alsoanticipated that these morphogens are capable of inducingredifferentiation of committed cells under appropriate environmentalconditions.

In one aspect, the morphogens of this invention comprise amorphogenically active dimeric species comprising a pair of polypeptidechains, wherein at least one of the polypeptide chains comprises theamino acid sequence defined by residues 303 to 399 of Seq. ID No. 1including allelic, species and other mutant variants thereof. Inpreferred morphogens, at least one polypeptide chain comprises thesequence defined by residues 298-399, residues 261-399 or residues264≧399 of Seq. ID No. 1. Alternatively, the amino acid sequence of bothpolypeptide chains may be defined by part or all of the amino acidsequence of Seq. ID No. 1, including allelic, species and other mutantvariants thereof including chimeric constructs as described below. Whereonly one polypeptide chain is defined by the amino acid sequence of partor all of Seq. ID. No. 1, the other polypeptide chain preferablycomprises at least the sequence defining the C-terminal six cysteineskeleton of any of the other known morphogen family members, includingOP-1, OP-2, CBMP2A, CBMP2B, BMP3, BMP5, BMP6, Vgr-1, Vgl, 60A, DPP andGDF-1 (described, for example, in U.S. Ser. No. 752,764, abandoned,923,780, abandoned, or U.S. 92/07358), including allelic, species andother mutant variants thereof, including chimeric variants. Other usefulsequences include biosynthetic constructs, such as are described in U.S.Pat. No. 5,011,691.

In still another aspect of the invention, generic sequences are providedwhich accommodate the sequence identity of useful morphogens andincorporate OP-3's novel features.

In another aspect of the invention, morphogens of this inventioncomprise morphogenically active proteins encoded by part or all of thegenetic sequence listed in Seq. ID No. 1, including allelic, species andother mutant variants thereof. In still another aspect, the inventioncomprises morphogens encoded by nucleic acids that hybridize to part orall of the pro region of the OP-3 protein, bases 120 to 848 of Seq IDNo. 1, under stringent hybridization conditions. As used herein,"stringent hybridization conditions" are defined as hybridization in 40%formamide, 5× SSPE, 5× Denhardt's Solution, and 0.1% SDS at 37° C.overnight, and washing in 0.1× SSPE, 0.1% SDS at 50° C.

In one aspect of the invention, morphogenically active fragments of OP-3are useful in the replacement of diseased or damaged tissue in a mammal,including, but not limited to, damaged lung tissue resulting fromemphysema; cirrhotic tissue, including cirrhotic kidney or liver tissue;damaged heart or blood vessel tissue, as may result fromcardiomyopathies and/or atherothrombotic or cardioembolic strokes;damaged stomach and other mucosal tissues of the gastrointestinal tractresulting from ulceric perforations and/or their repair; damaged nervetissue as may result from physical injury, degenerative diseases such asAlzheimer's disease, multiple sclerosis, or strokes; damaged cartilageand bone tissue as may result from metabolic bone diseases and otherbone remodeling disorders; damaged dentin, periodontal and/or cementumtissue as may result from disease or mechanical injury; and in thereplacement of damaged tissue as a result of inflammation and/or chronicinflammatory disease.

As provided herein, morphogenically active fragments of OP-3 areprovided to a tissue-specific locus in vivo, to induce the developmentalcascade of tissue morphogenesis at that site. Cells stimulated ex vivoby contact with OP-3 also may be provided to the tissue locus. In thesecases the existing tissue provides the necessary matrix requirements,providing a suitable substratum or scaffold for the proliferating anddifferentiating cells in a morphogenically permissive environment, aswell as providing the necessary signals for directing thetissue-specificity of the developing tissue. The proteins or stimulatedcells also may be combined with a formulated matrix and implanted as adevice at a locus in vivo. The formulated matrix should be abiocompatible, preferably biodegradable acellular matrix having thecharacteristics described below. Where the necessary signals fordirecting the tissue-specificity of the developing tissue are notprovided endogenously, the matrix preferably also is tissue-specific.

In another aspect, the members of the morphogen protein family also cancontrol the body's cellular and humoral inflammatory response to aforeign object or an initial tissue injury. In many instances, the lossof tissue function results from the tissue destructive effects and thesubsequent formation of scar tissue associated with the body'simmune/inflammatory response to an initial or repeated injury to thetissue. The degree of scar tissue formation generally depends on theregenerative properties of the injured tissue, and on the degree andtype of tissue damage. Thus, in another aspect, morphogenically activefragments of OP-3 may be used to prevent or to substantially inhibit theformation of scar tissue, including alleviating immune response-mediatedtissue damage, by providing OP-3 or cells stimulated by exposure to OP-3protein, to a newly injured tissue locus. The OP-3 protein also may beprovided as a prophylactic, provided to a site in anticipation of tissueinjury, such as part of a surgical or other clinical procedure likely toproduce tissue damage, and to induce an inflammatory/immune response. Ina particularly useful embodiment, OP-3 may be used as part of atransplant procedure, to enhance the tissue viability of the organand/or tissue to be transplanted. The morphogen may be provided to theorgan and/or tissue to be transplanted prior to harvest, during itstransport, and/or during transplantation into the recipient host asdescribed below.

OP-3 also may be used to increase or regenerate a mesenchymal progenitoror stem cell population in a mammal. For example, progenitor cells maybe isolated from an individual's bone marrow, stimulated ex vivo withmorphogenic OP-3 for a time and at a concentration sufficient to inducethe cells to proliferate, and returned to the bone marrow. Other sourcesof progenitor cells that may be suitable include biocompatible cellsobtained from a cultured cell line, stimulated in culture, andsubsequently provided to the body. Alternatively, OP-3 may be providedby systemic (e.g., oral or parenteral) administration, or it may beinjected or otherwise provided to a progenitor cell population in anindividual to induce its mitogenic activity in vivo. For example, amorphogenically active fragment of OP-3 may be provided to the cells invivo, e.g., by systemic injection, to induce mitogenic activity.Similarly, a particular population of hemopoietic stem cells may beincreased by exposure to OP-3, for example by perfusing(plasmaphoresing) an individual's blood to extract the cells ofinterest, stimulating these cells ex vivo, and returning the stimulatedcells to the blood.

It is anticipated that the ability to augment an individual's progenitorcell population will enhance existing methods for treating disordersresulting from a loss or reduction of a renewable cell populationsignificantly. Two particularly significant applications include thetreatment of blood disorders and diseases involving impaired or lostimmune function.

The morphogens of this invention also can inhibit proliferation ofepithelial cell populations. The ability to inhibit epithelial cellproliferation may be exploited to reduce tissue damage associated withpsoriasis and dermatitis, and other inflammatory skin diseases, as wellas ulcerative diseases of the gastrointestinal tract, such as, forexample, in the healing of ulcers, including gastric ulcers, and theulcerations induced in oral mucocitis and inflammatory bowel disease.Morphogens may be used to particular advantage as a cytoprotective agentin clinical therapies likely to effect proliferating epithelialpopulations, such as cancer radiotherapies and chemotherapies thattypically induce oral mucositis, hair loss and/or skin disorders.

In another aspect of the invention, morphogenic OP-3 may be used tosupport the growth and maintenance of differentiated cells, inducingexisting differentiated cells to continue expressing their phenotype. Itis anticipated that this activity will be particularly useful in thetreatment of tissue disorders where loss of function is caused byreduced or lost metabolic function in which cells become senescent orquiescent, such as may occur in aging cells and/or may be manifested inosteoporosis and a number of nerve degenerative diseases, includingAlzheimer's disease. Application of OP-3 directly to the cells to betreated, or providing it systemically, as by oral or parenteraladministration, can stimulate these cells to continue expressing theirphenotype, thereby significantly reversing the effects of thedysfunction. In addition, a morphogenically active fragment of OP-3 alsomay be used in gene therapy protocols to stimulate the growth ofquiescent cells, thereby potentially enhancing the ability of thesecells to incorporate exogenous DNA.

In yet another aspect of the invention, a morphogenically activefragment of OP-3 also may be used to induce "redifferentiation" of cellsthat have strayed from their differentiation pathway, such as can occurduring tumorgenesis. It is anticipated that this activity will beparticularly useful in treatments to reduce or substantially inhibit thegrowth of neoplasms. The method also is anticipated to induce the de-and/or re-differentiation of these cells. As described supra, amorphogenically active OP-3 fragment may be provided to the cellsdirectly or systemically, stimulating these cells to revert back to amorphology and phenotype characteristic of untransformed cells.

In still another aspect of the invention, OP-3 may be used to stimulatecell adhesion molecule (CAM) expression levels in a cell. CAMs aremolecules defined as carrying out cell-cell interactions necessary fortissue formation. CAMs are believed to play a fundamental regulatoryrole in tissue development, including tissue boundary formation,embryonic induction and migration, and tissue stabilization andregeneration. Altered CAM levels have been implicated in a number oftissue disorders, including congenital defects, neoplasias, anddegenerative diseases.

In particular, N-CAM expression is associated with normal neuronal celldevelopment and differentiation, including retinal formation,synaptogenesis, and nerve-muscle tissue adhesion. Inhibition of one ormore of the N-CAM isoforms is known to prevent proper tissuedevelopment. Altered N-CAM expression levels also are associated withneoplasias, including neuroblastomas (see infra), as well as with anumber of neuropathies, including normal pressure hydrocephalous andtype II schizophrenia. Application of the morphogen directly to thecells to be treated, or providing the morphogen to the mammalsystemically, for example, parenterally, or indirectly by oraladministration, may be used to induce cellular expression of one or moreCAMs, particularly N-CAMs and L1.

CAMs also have been postulated as part of a morphoregulatory pathwaywhose activity is induced by a to date unidentified molecule (See, forexample, Edelman, G. M. (1986) Ann. Rev. Cell Biol., 2:81-116). Withoutbeing limited to any given theory, the morphogens described herein mayact as inducers of this pathway.

The matrices utilized in the methods of the invention may be derivedfrom organ-specific tissue, or they may be formulated synthetically. Inone embodiment of the invention, when OP-3 (or a collection ofprogenitor cells stimulated by OP-3) is provided at a tissue-specificlocus, e.g., by systemic administration, implantation or injection at atissue-specific locus, the existing tissue at that locus, whetherdiseased or damaged, has the capacity of acting as a suitable matrix orscaffold for the differentiation and proliferation of migratingprogenitor cells. Alternatively, a formulated matrix may be providedexternally together with the stimulated progenitor cells ormorphogenically active OP-3 fragment, as may be necessary when theextent of injury sustained by the damaged tissue is large. The matrixshould be a biocompatible, suitably modified acellular matrix havingdimensions such that it allows the differentiation and proliferation ofmigratory progenitor cells, and is capable of providing amorphogenically permissive environment. The matrix also preferablyallows cellular attachment and is biodegradable. Where the necessarytissue-directing signals can not be provided endogenously, the matrixpreferably also is tissue-specific.

Formulated matrices may be generated from dehydrated organ-specifictissue prepared, for example, by treating the tissue with solvents tosubstantially remove the intracellular, non-structural components fromthe tissue. Alternatively, the matrix may be formulated syntheticallyusing a biocompatible, preferably in vivo biodegradable, structuralmolecule, and may be formulated with suitable tissue-specific cellattachment factors. The molecule may be a naturally occurring one suchas collagen, laminin or hyaluronic acid, or a synthetic polymercomprising, for example, polylactic acid, polybutyric acid, polyglycolicacid, and copolymers thereof. Currently preferred structural polymerscomprise tissue-specific collagens. Currently preferred cell attachmentfactors include glycosaminoglycans and proteoglycans. The matrix furthermay be treated with an agent or agents to increase the number of poresand micropits on its surfaces, so as to enhance the influx,proliferation and differentiation of migratory progenitor cells from thebody of the mammal.

The invention thus relates to compositions and methods for the use ofmorphogenically active fragments of OP-3, a novel species variant of thegeneric family of morphogens disclosed in U.S. Ser. No. 667,274,abandoned, and U.S. Ser. No. 752,764, abandoned, as a tissue morphogen.Morphogenically active OP-3 and protein fragments can be isolated fromnaturally-occurring sources, or they may be constructed biosyntheticallyusing conventional recombinant DNA technology. Active OP-3 useful in thecompositions and methods of this invention may include forms havingvarying glycosylation patterns, varying N-termini and active truncatedforms, e.g., produced by recombinant DNA techniques. Active OP-3proteins also include chimeric constructs as described below, comprisingboth an OP-3 active domain and a non-OP-3 sequence as, for example, thepro domain and/or the N-terminal region of the mature protein. OP-3protein can be expressed from intact or truncated cDNA or from syntheticDNAs in procaryotic or eucaryotic host cells, and purified, cleaved,refolded, and dimerized to form morphogenically active compositions.Useful host cells include procaryotes, including E. coli, and eucaryoticcells, including mammalian cells, such as CHO, COS, melanoma or BSCcells, or the insect/baculovirus system. Thus recombinant DNA techniquesmay be utilized to produce large quantities of OP-3 capable of inducingtissue-specific cell differentiation and tissue morphogenesis in avariety of mammals, including humans.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a nucleotide sequence comparison of the mouse cDNA sequence ofOP-2 and OP-3. Exon boundaries are indicated by bars beneath thesequence; diamonds indicate nucleotide differences within exons 2 and 3.

DETAILED DESCRIPTION

The invention provides a novel genetic sequence, mOP-3, encoding a novelprotein, OP-3, having morphogenic properties. The genetic sequenceoriginally was identified in a mouse cDNA library, and the inventionprovides methods for identifying and isolating the gene from otherspecies. As will be appreciated by those skilled in the art, the methodsdescribed herein also may be used to isolate the OP-3 gene from otherlibraries, including genomic libraries. The invention also providesmeans for producing the OP-3 genetic sequence and the encoded protein.The invention further provides methods and compositions for inducing thedevelopmental cascade of tissue morphogenesis in a mammal utilizingmorphogenically active fragments of OP-3. The methods and compositionsprovided herein may be utilized in a range of applications, includingstimulating the proliferation and/or differentiation of progenitor cellsand inducing the repair and regeneration of damaged tissue. Themorphogenic OP-3 species of the invention are novel species variants ofthe family of morphogens disclosed in copending U.S. Ser. No. 667,274,abandoned, U.S. Ser. No. 752,764, abandoned, U.S. Ser. No. 923,780,abandoned, and U.S. Ser. No. 922,813, abandoned, the disclosures ofwhich are incorporated hereinabove by reference. As described herein,OP-3 may be isolated from natural sources or constructedbiosynthetically utilizing conventional recombinant DNA technology orconstructed synthetically using standard chemical techniques.

Morphogenically active fragments of OP-3 are useful for initiating andmaintaining the tissue-specific developmental cascade in a variety oftissues, including, but not limited to, bone, cartilage, dentin, neuraltissue, liver, periodontal ligament, cementum, lung, heart, kidney andnumerous tissues of the gastrointestinal tract. When combined with naivemesenchymal progenitor cells as disclosed herein, OP-3 can induce theproliferation and differentiation of these progenitor cells. In thepresence of appropriate tissue-specific signals to direct thedifferentiation of these cells, and a morphogenically permissiveenvironment, OP-3 is capable of reproducing the cascade of cellular andmolecular events that occur during embryonic development to yieldfunctional tissue. For example, the protein can induce the de novoformation of cartilage and endochondral bone, including inducing theproliferation and differentiation of progenitor cells into chondrocytesand osteoblasts, inducing appropriate mineralization and boneremodeling, inducing formation of an appropriate bone tissue vascularsupply and inducing formation of differentiated bone marrow (see Example7 below.)

Provided below is a detailed description of the nucleic acid and aminoacid sequences which describe OP-3 proteins useful in the compositionsand methods of this invention, including a description of how to makethem, and methods and means for their therapeutic administration. Alsoprovided are numerous, nonlimiting examples which (1) illustrate thesuitability of these proteins as tissue morphogens and therapeuticagents, and (2) provide assays with which to test the morphogensencompassed by the invention in different tissues. Also provided inExample 9 is a method for screening compounds to identify morphogenstimulating agents capable of stimulating endogenous OP-3 expressionand/or secretion. OP-3 stimulating agents then may be used in any of thetherapeutic applications described herein in place of, or in additionto, OP-3 protein administration.

I. Useful Morphogens

As defined herein a protein is morphogenic if it is capable of inducingthe developmental cascade of cellular and molecular events thatculminate in the formation of new, organ-specific tissue and comprisesat least the conserved C-terminal six cysteine skeleton or itsfunctional equivalent (see supra). Specifically, the morphogensgenerally are capable of all of the following biological functions in amorphogenically permissive environment: stimulating proliferation ofprogenitor cells; stimulating the differentiation of progenitor cells;stimulating the proliferation of differentiated cells; and supportingthe growth and maintenance of differentiated cells, including the"redifferentiation" of transformed cells. Details of how the morphogenfamily of proteins described herein first were identified, as well as adescription of how to make, use and test them for morphogenic activityare disclosed in U.S. Ser. No. 667,274, abandoned, filed Mar. 11, 1991and U.S. Ser. No. 752,764, abandoned, filed Aug. 30, 1991. As disclosedtherein, the morphogens may be purified from naturally-sourced materialor recombinantly produced from procaryotic or eucaryotic host cells,preferably as described therein. Alternatively, novel morphogenicsequences may be identified following the procedures disclosed therein.

Particularly useful morphogens identified to date include OP-1, OP-2,CBMP2A and CBMP2B (the morphogenically active domains of proteinsreferred to in the art as BMP2A and BMP2B, or BMP2 and BMP4,respectively), BMP3, BMP5, BMP6, Vgr-1, GDF-1, Vgl, DPP and 60A,including their allelic and species variants, as well as other mutantvariants including chimeric morphogens. Detailed descriptions of theproteins may be found in, for example, U.S. Ser. No. 752,764, abandoned,or U.S. Ser. No. 922,813, abandoned. Morphogenically active biosyntheticconstructs such as those disclosed in U.S. Pat. No. 5,011,691, thedisclosure of which is incorporated herein by reference (e.g., COP-1,COP-3, COP-4, COP-5, COP-7, and COP-16) also are envisioned to beuseful.

Using the methodology disclosed in U.S. Ser. No. 752,764, abandoned, andU.S. Ser. No. 667,274, abandoned, the novel morphogen OP-3 and itsgenetic sequence, now have been identified. The OP-3 proteins useful inthe invention include any morphogenically active fragment of the OP-3amino acid sequence present in Seq. ID No. 1, or allelic, species orother mutant variants thereof. The morphogenically active fragment ofOP-3 also may include any morphogenically active protein encoded by partor all of the nucleic acid sequence presented in Seq. ID No. 1. Themorphogenic protein also may comprise a protein encoded by part or allof a nucleic acid which hybridizes to at least part of the nucleic acidsequence encoding the "pro" region of the OP-3 protein, bases 120-848 ofSeq. ID No. 1, under stringent conditions.

The mOP-3 gene encodes a protein ("mOP-3") first expressed as animmature translation product that is 399 amino acids in length. Thisprecursor form, referred to herein as the "prepro" form, (Seq. ID. No.1, amino acid residues 1-399) includes an N-terminal signal peptidesequence, typically less than about 20 residues, followed by a "pro"domain that is cleaved to yield the mature sequence. The "pro" form ofthe protein includes the pro domain and the mature domain, and forms asoluble species that appears to be the primary form secreted fromcultured mammalian cells. The signal peptide is cleaved rapidly upontranslation, at a cleavage site that can be predicted in a givensequence using the method of Von Heijne ((1986) Nucleic Acids Research14:4683-4691). The preferred form of morphogenically active OP-3 proteincomprises a processed sequence, including fragments thereof,appropriately dimerized and disulfide bonded. Where a soluble form ofthe protein is desired, the protein preferably comprises both the maturedomain, or an active portion thereof, and part or all of the pro domain.

By amino acid sequence homology with other, known morphogens, the prodomain likely is cleaved at residues 257-260 of Seq. ID No. 1, whichrepresent the canonical Arg-Xaa-Xaa-Arg cleavage site, to yield a maturesequence 139 amino acids in length (Seq. ID No. 1, residues 261-399).Alternatively, the pro domain may be cleaved at residues 260-263 toyield a shorter sequence 135 amino acids in length (Seq. ID No. 1, aminoacid residues 264-399). All morphogens, including OP-1, OP-2 and theOP-3 proteins disclosed herein, comprise at least a conserved sixcysteine skeleton in the amino acid sequence C-terminal domain and,preferably, a conserved seven cysteine skeleton (see, for example, U.S.Ser. No. 752,764, abandoned). The conserved six cysteine skeleton inmOP-3 (Seq. ID No. 1) is defined by amino acid residues 303-399;theconserved seven cysteine skeleton is defined by amino acid residues298-399. In addition to the conserved six cysteine skeleton found inknown morphogen family members including OP-1, OP-2, CBMP2A, CBMP2B,BMP3, BMP5, BMP6, Vgr-1, Vgl, 60A, DPP and GDF-1, (described, forexample, in U.S. Ser. No. 752,764 and 923,780), the OP-3 proteins, likethe OP-2 proteins, also has one additional cysteine residue (residue 338of Seq. ID No. 1) in the conserved C-terminal domain.

The mature sequence of OP-3 shares significant amino acid sequencehomology with the morphogens identified to date. Specifically, the sevencysteine fragment shows greater than 79% amino acid identity with thecorresponding mOP-2 and hOP-2 sequences, and greater than 66% identitywith the corresponding OP-1 sequences. Like OP-2, OP-3 has an eighthcysteine within the seven cysteine domain (e.g., at position 338 of Seq.ID No. 1). In addition, OP-3 is unique among the morphogens identifiedto date in that the residue at position 9 in the conserved sevencysteine domain (e.g., residue 315 of Seq. ID No. 1) is a serine,whereas other morphogens typically have a tryptophan at this location(see Table I below, and Table II in PCT/US92/07358, for example.)

Thus, useful OP-3 mutant variants include, but are not limited to, aminoacid sequences derived from Seq. ID No. 1 and wherein the cysteine atposition 338 is replaced with another amino acid, preferably a tyrosine,histidine, isoleucine or serine and conservative substitutions thereof,e.g., such as defined by Dayoff et al., Atlas of Protein Sequence andStructure; vol. 5, Suppl. 3, pp. 345-362 (M. O. Dayoff, ed., Nat'lBioMed. Research Fdn., Washington D.C. 1979.). Still other useful OP-3mutant variants include proteins wherein the serine at position 315 isreplaced with another amino acid, preferably a tryptophan andconservative substitutions thereof.

Generic Sequence 7 (Seq. ID No. 12) and Generic Sequence 8 (Seq. ID No.13) disclosed below, accommodate the homologies shared among preferredmorphogen protein family members identified to date, including OP-1,OP-2, OP-3, CBMP2A, CBMP2B, BMP3, 60A, DPP, Vgl, BMP5, BMP6, Vrg-1, andGDF-1. The amino acid sequences for these proteins are described herein(see Sequence Listing and Table I below) and/or in the art, as well asin PCT publication U.S. Ser. No. 92/07358, filed Aug. 28, 1992, forexample. The generic sequences include both the amino acid identityshared by these sequences in the C-terminal domain, defined by the sixand seven cysteine skeletons (Generic Sequences 7 and 8, respectively),as well as alternative residues for the variable positions within thesequence. The generic sequences allow for an additional cysteine atposition 41 (Generic Sequence 7) or position 46 (Generic Sequence 8),providing an appropriate cysteine skeleton where inter- orintramolecular disulfide bonds can form, and containing certain criticalamino acids which influence the tertiary structure of the proteins.##STR1## wherein each Xaa is independently selected from a group of oneor more specified amino acids defined as follows: "Res." means "residue"and Xaa at res.2=(Tyr or Lys); Xaa at res.3=Val or Ile); Xaa atres.4=(Ser, Asp or Glu); Xaa at res.6=(Arg, Gln, Ser, Lys or Ala); Xaaat res.7=(Asp or Glu); Xaa at res.8=(Leu, Val or Ile); Xaa atres.11=(Gln, Leu, Asp, His, Asn or Ser); Xaa at res.12=(Asp, Arg, Asn orGlu); Xaa at res.13=(Trp or Ser); Xaa at res.14=(Ile or Val); Xaa atres.15=(Ile or Val); Xaa at res.16 (Ala or Ser); Xaa at res.18=(Glu,Gln, Leu, Lys, Pro or Arg); Xaa at res.19=(Gly or Ser); Xaa atres.20=(Tyr or Phe); Xaa at res.21=(Ala, Ser, Asp, Met, His, Gln, Leu orGly); Xaa at res.23=(Tyr, Asn or Phe); Xaa at res.26=(Glu, His, Tyr,Asp, Gln, Ala or Ser); Xaa at res.28=(Glu, Lys, Asp, Gln or Ala); Xaa atres.30=(Ala, Ser, Pro, Gln, Ile or Asn); Xaa at res.31=(Phe, Leu orTyr); Xaa at res.33=(Leu, Val or Met); Xaa at res.34=(Asn, Asp, Ala, Thror Pro); Xaa at res.35=(Ser, Asp, Glu, Leu, Ala or Lys); Xaa atres.36=(Tyr, Cys, His, Ser or Ile); Xaa at res.37=(Met, Phe, Gly orLeu); Xaa at res.38=(Ash, Ser or Lys); Xaa at res.39=(Ala, Ser, Gly orPro); Xaa at res.40=(Thr, Leu or Ser); Xaa at res.44=(Ile, Val or Thr);Xaa at res.45=(Val, Leu, Met or Ile); Xaa at res.46=(Gln or Arg); Xaa atres.47=(Thr, Ala or Ser); Xaa at res.48=(Leu or Ile); Xaa at res.49=(Valor Met); Xaa at res.50=(His, Asn or Arg); Xaa at res.51=(Phe, Leu, Asn,Ser, Ala or Val); Xaa at res.52=(Ile, Met, Asn, Ala, Val, Gly or Leu);Xaa at res.53=(Asn, Lys, Ala, Glu, Gly or Phe); Xaa at res.54=(Pro, Seror Val); Xaa at res.55=(Glu, Asp, Asn, Gly, Val, Pro or Lys); Xaa atres.56=(Thr, Ala, Val, Lys, Asp, Tyr, Ser, Gly, Ile or His); Xaa atres.57=(Val, Ala or Ile); Xaa at res.58=(Pro or Asp); Xaa atres.59=(Lys, Leu or Glu); Xaa at res.60=(Pro, Val or Ala); Xaa atres.63=(Ala or Val); Xaa at res.65=(Thr, Ala or Glu); Xaa atres.66=(Gln, Lys, Arg or Glu); Xaa at res.67=(Leu, Met or Val); Xaa atres.68=(Asn, Ser, Asp or Gly); Xaa at res.69=(Ala, Pro or Ser); Xaa atres.70=(Ile, Thr, Val or Leu); Xaa at res.71=(Ser, Ala or Pro); Xaa atres.72=(Val, Leu, Met or Ile); Xaa at res.74=(Tyr or Phe); Xaa atres.75=(Phe, Tyr, Leu or His); Xaa at res.76=(Asp, Asn or Leu); Xaa atres.77=(Asp, Glu, Asn, Arg or Ser); Xaa at res.78=(Ser, Gln, Asn, Tyr orAsp); Xaa at res.79=(Ser, Asn, Asp, Glu or Lys); Xaa at res.80=(Asn, Thror Lys); Xaa at res.82=(Ile, Val or Asn); Xaa at res.84=(Lys or Arg);Xaa at res.85=(Lys, Asn, Gln, His, Arg or Val); Xaa at res.86=(Tyr, Gluor His); Xaa at res.87=(Arg, Gln, Glu or Pro); Xaa at res.88=(Ash, Glu,Trp or Asp); Xaa at res.90=(Val, Thr, Ala or Ile); Xaa at res.92=(Arg,Lys, Val, Asp, Gln or Glu); Xaa at res.93=(Ala, Gly, Glu or Ser); Xaa atres.95=(Gly or Ala) and Xaa at res.97=(His or Arg).

As described above, Generic Sequence 8 (Seq. ID No. 13) includes all ofGeneric Sequence 7 and in addition includes the following sequence atits N-terminus: ##STR2##

Accordingly, beginning with residue 7, each "Xaa" in Generic Seq. 8 is aspecified amino acid defined as for Generic Seq. 7, with the distinctionthat each residue number described for Generic Sequence 7 is shifted byfive in Generic Seq. 8. Thus, "Xaa at res.2=(Tyr or Lys)" in Gen. Seq. 7refers to Xaa at res. 7 in Generic Seq. 8. In Generic Seq. 8, Xaa atres.2=(Lys, Arg, Ala or Gln); Xaa at res.3=(Lys, Arg or Met); Xaa atres.4=(His, Arg or Gln); and Xaa at res.5=(Glu, Ser, His, Gly, Arg, Pro,Thr, or Tyr).

Table I, set forth below, compares the C-terminal amino acid sequencesdefining the seven cysteine skeleton of human OP-1, mouse OP-1, humanOP-2, mouse OP-2, and mouse OP-3 (mOP-3, Seq. ID No. 1). In the table,the sequences are aligned essentially following the method of Needlemanet al. (1970) J. Mol. Biol., 48:443-453, calculated using the AlignProgram (DNAstar, Inc.) In the table, three dots indicates that theamino acid in that position is the same as the amino acid in hOP-1.Three dashes indicate that no amino acid is present in that position,and are included for purposes of illustrating homologies. As is apparentfrom the following amino acid sequence comparisons, significant aminoacid sequence homology exists between mouse OP-3 and mouse and humanOP-1 and OP-2.

                                      TABLE I    __________________________________________________________________________    Seq.    ID    No.    __________________________________________________________________________    hOP-1        3    Cys                Lys                   Lys                      His                         Glu                            Leu                               Try                                  Val                                     Ser                                        Phe                                           Arg                                              Asp                                                 Lue                                                    Gly                                                       Trp                                                          Gln                                                             Asp    mOP-1        5    -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --    hOP-2        7    -- Arg                   Arg                      -- -- -- -- -- -- -- Gln                                              -- -- -- -- Leu                                                             --    mOP-2        9    -- Arg                   Arg                      -- -- -- -- -- -- -- -- -- -- -- -- Leu                                                             --    mOP-3        1    -- Arg                   Arg                      -- -- -- -- -- -- -- -- -- -- -- -- Leu                                                             --             1           5           10             15    __________________________________________________________________________    hOP-1    Trp                Ile                   Ile                      Ala                         Pro                            Glu                               Gly                                  Try                                     Ala                                        Ala                                           Tyr                                              Tyr                                                 Cys                                                    Glu                                                       Gly                                                          Glu                                                             Cys Ala    mOP-1    -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --  --    hOP-2    -- Val                   -- -- -- Gln                               -- -- Ser                                        -- -- -- -- -- -- -- Ser --    mOP-2    -- Val                   -- -- -- Gln                               -- -- Ser                                        -- -- -- -- -- -- -- --  --    mOP-3    Ser                Val                   -- -- -- Gln                               -- -- Ser                                        -- -- -- -- Ala                                                       -- -- --  Ile                20                25             30              35    __________________________________________________________________________    hOP-1    Phe                Pro                   Leu                      Asn                         Ser                            Tyr                               Met                                  Asn                                     Ala                                        Thr                                           Asn                                              His                                                 Ala                                                    Ile                                                       Val                                                          Gln                                                             Thr Leu    mOP-1    -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --  --    hOP-2    -- -- -- Asp                         -- Cys                               -- -- -- -- -- -- -- -- Leu                                                          -- Ser --    mOP-2    -- -- -- Asp                         -- Cys                               -- -- -- -- -- -- -- -- Leu                                                          -- Ser --    mOP-3    Tyr                -- -- -- -- Cys                               -- -- Ser                                        -- -- -- -- Thr                                                       Met                                                          -- Ala --                         40             45             50    __________________________________________________________________________    hOP-1    Val                His                   Phe                      Ile                         Asn                            Pro                               Glu                                  Thr                                     Val                                        Pro                                           Lys                                              Pro                                                 Cys                                                    Cys                                                       Ala                                                          Pro                                                             Thr Gln    mOP-1    -- -- -- -- -- -- Asp                                  -- -- -- -- -- -- -- -- -- --  --    hOP-2    -- -- Leu                      Met                         Lys                            -- Asn                                  Ala                                     -- -- -- Ala                                                 -- -- -- -- --  Lys    mOP-2    -- -- Leu                      Met                         Lys                            -- Asp                                  Val                                     -- -- -- Ala                                                 -- -- -- -- --  Lys    mOP-3    -- -- Leu                      Met                         Lys                            -- Asp                                  Ile                                     Ile                                        -- -- Val                                                 -- -- Val                                                          -- --  Glu                55             60             65             70    __________________________________________________________________________    hOP-1    Lue                Asn                   Ala                      Ile                         Ser                            Val                               Leu                                  Tyr                                     Phe                                        Asp                                           Asp                                              Ser                                                 Ser                                                    Asn                                                       Val                                                          Ile                                                             Leu Lys    mOP-1    -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --  --    hOP-2    -- Ser                   -- Thr                         -- -- -- -- Tyr                                        -- Ser                                              -- Asn                                                    -- -- -- --  Arg    mOP-2    -- Ser                   -- Thr                         -- -- -- -- Tyr                                        -- Ser                                              -- Asn                                                    -- -- -- --  Arg    mOP-3    -- Ser                   -- -- -- Leu                               -- -- Tyr                                        -- Arg                                              Asn                                                 Asn                                                    -- -- -- --  Arg                      75             80             85    __________________________________________________________________________    hOP-1    Lys                Tyr                   Arg                      Asn                         Met                            Val                               Val                                  Arg                                     Ala                                        Cys                                           Gly                                              Cys                                                 His    mOP-1    -- -- -- -- -- -- -- -- -- -- -- -- --    hOP-2    -- His                   -- -- -- -- -- Lys                                     -- -- -- -- --    mOP-2    -- His                   -- -- -- -- -- Lys                                     -- -- -- -- --    mOP-3    Arg                Glu                   -- -- -- -- -- Gln                                     -- -- -- -- --             90             95             100    __________________________________________________________________________

physiologic saline (0.9% NaCl, 0.15M), pH 7-7.4. The aqueous solutioncontaining the morphogen can be made, for example, by dissolving theprotein in 50% ethanol, or acetonitrile containing 0.1% trifluoroaceticacid (TFA) or 0.1% HCl, or equivalent solvents. One volume of theresultant solution then is added, for example, to ten volumes ofphosphate buffered saline (PBS), which further may include 0.1-0.2%human serum albumin (HSA). The resultant solution preferably is vortexedextensively.

If desired, a given morphogen may be made more soluble by associationwith a suitable molecule. For example, association of the mature dimerwith the pro domain of the morphogen increases solubility of the proteinsignificantly. For example, the pro form of OP-3 comprises a speciesthat is soluble in physiologically buffered solutions. In fact, theendogenous protein is thought to be transported (e.g., secreted andcirculated) to particular tissues in this form. This soluble form of theprotein may be obtained from the culture medium of morphogen-secretingmammalian cells. Alternatively, a soluble species may be formulated bycomplexing the mature dimer (or an active fragment thereof) with part orall of a pro domain. Another molecule capable of enhancing solubilityand particularly useful for oral administrations, is casein. Forexample, addition of 0.2% casein increases solubility of the matureactive form of OP-1 by 80%. Other components found in milk and/orvarious serum proteins also may be useful.

Useful solutions for oral or parenteral administration may be preparedby any of the methods well known in the pharmaceutical art, described,for example, in Remington's Pharmaceutical Sciences, (Gennaro, A., ed.),Mack Pub., 1990. Formulations may include, for example, polyalkyleneglycols such as polyethylene glycol, oils of vegetable origin,hydrogenated naphthalenes, and the like. Formulations for directadministration, in particular, may include glycerol and othercompositions of high viscosity. Biocompatible, preferably bioresorbablepolymers, including, for example, hyaluronic acid, collagen, tricalciumphosphate, polybutyrate, polylactide, polyglycolide andlactide/glycolide copolymers, may be useful excipients to control therelease of the morphogen in vivo.

Other potentially useful parenteral delivery systems for thesemorphogens include ethylene-vinyl acetate copolymer particles, osmoticpumps, implantable infusion systems, and liposomes. Formulations forinhalation administration may contain as excipients, for example,lactose, or may be aqueous solutions containing, for example,polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oilysolutions for administration in the form of nasal drops, or as a gel tobe applied intranasally. Formulations for parenteral administration mayalso include glycocholate for buccal administration, methoxysalicylatefor rectal administration, or cutric acid for vaginal administration.

Alternatively, the morphogens described herein may be administeredorally. Oral administration of proteins as therapeutics generally is notpracticed as most proteins readily are degraded by digestive enzymes andacids in the mammalian digestive system before they can be absorbed intothe bloodstream. However, the morphogens described herein typically areacid-stable and protease-resistant (see, for example, U.S. Pat. No.4,968,590.) In addition, at least one morphogen, OP-1, has beenidentified in bovine mammary gland extract, colostrum and milk, as wellas saliva. Moreover, the OP-1 purified from mammary gland extract ismorphogenically active. For example, this protein induces endochondralbone formation in mammals when implanted subcutaneously in associationwith a suitable matrix material, using a standard in vivo bone assay,such as is disclosed in U.S. Pat. No. 4,968,590. In addition, endogenousmorphogen also is detected in human serum. These findings indicate thatoral and parenteral administration are viable means for administeringmorphogens to an individual. Moreover, while the mature forms of certainmorphogens described herein typically are sparingly soluble, themorphogen form found in milk (and mammary gland extract and colostrum)is readily soluble, probably by association of the mature,morphogenically active form with the pro domain of the intact sequenceand/or by association with one or more milk components. Accordingly, thecompounds provided herein also may be associated with molecules capableof enhancing their solubility in vitro or in vivo, including, forexample, part or all of a morphogen pro domain, and casein, as describedabove.

The compounds provided herein also may be associated with moleculescapable of targeting the morphogen to a desired tissue. For example,tetracycline and diphosphonates (bisphosphonates) are known to bind tobone mineral, particularly at zones of bone remodeling, when they areprovided systemically in a mammal. Accordingly, these molecules may beincluded as useful agents for targeting OP-3 to bone tissue.Alternatively, an antibody or other binding protein that interactsspecifically with a surface molecule on the desired target tissue cellsalso may be used. Such targeting molecules further may be covalentlyassociated to the morphogen, e.g., by chemical crosslinking, or by usingstandard genetic engineering means to create, for example, an acidlabile bond such as an Asp-Pro linkage. Useful targeting molecules maybe designed, for example, using the single chain binding site technologydisclosed, for example, in U.S. Pat. No. 5,091,513.

As described above, the morphogen family members share significantsequence homology in the C-terminal active domains. By contrast, thesequences diverge significantly in the sequences which define the prodomain and the N-terminal 39 amino acids of the mature protein.Accordingly, the pro domain and/or N-terminal sequence may bemorphogen-specific. As described above, it also is known that thevarious morphogens identified to date are differentially expressed inthe different tissues. Accordingly, without being limited to any giventheory, it is likely that, under natural conditions in the body,selected morphogens typically act on a given tissue. Accordingly, partor all of morphogen-specific sequences may serve as targeting moleculesfor the morphogens described herein. For example, the pro domains mayinteract specifically with one or more molecules at the target tissue todirect the morphogen associated with the pro domain to that tissue.Thus, another useful targeting molecule for targeting OP-3 to bonetissue, for example, may include part or all of a morphogen-specificsequence, such as part or all of a pro domain and/or the N-terminus ofthe mature protein. Particularly useful are the morphogen-specificsequences of OP-1, BMP2 or BMP4, all of which proteins are foundnaturally associated with bone tissue (see, for example, U.S. Pat. No.5,011,691). Alternatively, the morphogen-specific sequences of GDF-1 maybe used to target morphogenic OP-3 to nerve tissue, particularly braintissue where GDF-1 appears to be primarily expressed (see, for example,U.S. Ser. No. 922,813 and Lee, PNAS, 88:4250-4254 (1991), incorporatedherein by reference). As described above, pro forms of the proteins maybe obtained from the culture medium of morphogen-secreting mammaliancells. Alternatively, a suitable species may be formulated by complexingthe mature dimer (or an active fragment thereof) with part or all of apro domain. Chimeric OP-3 proteins comprising, for example, non-OP-3 prodomains and/or non-OP-3 N-termini, may be synthesized using standardrecombinant DNA methodology and/or automated chemical nucleic acidsynthesis methodology well described in the art and as disclosed below.

Finally, the OP-3 proteins provided herein may be administered alone orin combination with other molecules known to have a beneficial effect ontissue morphogenesis, including molecules capable of tissue repair andregeneration and/or inhibiting inflammation. Examples of usefulcofactors for stimulating bone tissue growth in osteoporoticindividuals, for example, include but are not limited to, vitamin D₃,calcitonin, prostaglandins, parathyroid hormone, dexamethasone, estrogenand IGF-I or IGF-II. Useful cofactors for nerve tissue repair andregeneration may include nerve growth factors. Other useful cofactorsinclude symptom-alleviating cofactors, including antiseptics,antibiotics, antiviral and antifungal agents and analgesics andanesthetics.

The compounds provided herein can be formulated into pharmaceuticalcompositions by admixture with pharmaceutically acceptable nontoxicexcipients and carriers. As noted above, such compositions may beprepared for parenteral administration, particularly in the form ofliquid solutions or suspensions; for oral administration, particularlyin the form of tablets or capsules; or intranasally, particularly in theform of powders, nasal drops or aerosols. Where adhesion to a tissuesurface is desired the composition may include the morphogen dispersedin a fibrinogen-thrombin composition or other bioadhesive such as isdisclosed, for example in PCT US91/09275, the disclosure of which isincorporated herein by reference. The composition then may be painted,sprayed or otherwise applied to the desired tissue surface.

The compositions can be formulated for parenteral or oral administrationto humans or other mammals in therapeutically effective amounts, e.g.,amounts which provide appropriate concentrations of OP-3 to targettissue for a time sufficient to induce morphogenesis, includingparticular steps thereof, as described above.

Where OP-3 is to be used as part of a transplant procedure, themorphogen may be provided to the living tissue or organ to betransplanted prior to removal of tissue or organ from the donor. OP-3may be provided to the donor host directly, as by injection of aformulation comprising OP-3 into the tissue, or indirectly, e.g., byoral or parenteral administration, using any of the means describedabove.

Alternatively or, in addition, once removed from the donor, the organ orliving tissue may be placed in a preservation solution containing OP-3.In addition, the recipient also preferably is provided with themorphogen just prior to, or concomitant with, transplantation. In allcases, OP-3 may be administered directly to the tissue at risk, as byinjection to the tissue, or it may be provided systemically, either byoral or parenteral administration, using any of the methods andformulations described herein and/or known in the art.

Where OP-3 comprises part of a tissue or organ preservation solution,any commercially available preservation solution may be used toadvantage. For example, useful solutions known in the art includeCollins solution, Wisconsin solution, Belzer solution, Eurocollinssolution and lactated Ringer's solution. Generally, an organpreservation solution usually possesses one or more of the followingproperties: (a) an osmotic pressure substantially equal to that of theinside of a mammalian cell, (solutions typically are hyperosmolar andhave K+ and/or Mg++ ions present in an amount sufficient to produce anosmotic pressure slightly higher than the inside of a mammalian cell);(b) the solution typically is capable of maintaining substantiallynormal ATP levels in the cells; and (c) the solution usually allowsoptimum maintenance of glucose metabolism in the cells. Organpreservation solutions also may contain anticoagulants, energy sourcessuch as glucose, fructose and other sugars, metabolites, heavy metalchelators, glycerol and other materials of high viscosity to enhancesurvival at low temperatures, free oxygen radical inhibiting and/orscavenging agents and a pH indicator. A detailed description ofpreservation solutions and useful components may be found, for example,in U.S. Pat. No. 5,002,965, the disclosure of which is incorporatedherein by reference.

OP-3 is envisioned to be useful in enhancing viability of any organ orliving tissue to be transplanted. The morphogens may be used toparticular advantage in lung, heart, liver, kidney or pancreastransplants, as well as in the transplantation and/or grafting of bonemarrow, skin, gastrointestinal mucosa, and other living tissues.

As will be appreciated by those skilled in the art, the concentration ofthe compounds described in a therapeutic composition will vary dependingupon a number of factors, including the dosage of the drug to beadministered, the chemical characteristics (e.g., hydrophobicity) of thecompounds employed, and the route of administration. The preferreddosage of drug to be administered also is likely to depend on suchvariables as the type and extent of tissue loss or defect, the overallhealth status of the particular patient, the relative biologicalefficacy of the compound selected, the formulation of the compound, thepresence and types of excipients in the formulation, and the route ofadministration. In general terms, the compounds of this invention may beprovided in an aqueous physiological buffer solution containing about0.001 to 10% w/v compound for parenteral administration. Typical doseranges are from about 10 ng/kg to about 1 g/kg of body weight per day; apreferred dose range is from about 0.1 μg/kg to 100 mg/kg of bodyweight. No obvious morphogen-induced pathological lesions are inducedwhen mature morphogen (e.g., OP-1, 20 μg) is administered daily tonormal growing rats for 21 consecutive days. Moreover, 10 μg systemicinjections of morphogen (e.g., OP-1) injected daily for 10 days intonormal newborn mice does not produce any gross abnormalities.

II.B Matrix Preparation

A morphogenically active fragment of OP-3 may be implanted surgically,dispersed in a biocompatible, preferably in vivo biodegradable matrixappropriately modified to provide a structure or scaffold in which theOP-3 may be dispersed and which allows the differentiation andproliferation of migrating progenitor cells. The matrix also may providesignals capable of directing the tissue specificity of thedifferentiating cells, as well as providing a morphogenically permissiveenvironment, being essentially free of growth inhibiting signals.

The formulated matrix may be shaped as desired in anticipation ofsurgery or may be shaped by the physician or technician during surgery.Thus, the material may be used in topical, subcutaneous,intraperitoneal, or intramuscular implants to repair tissue or to induceits growth de novo. The matrix preferably is biodegradable in vivo,being slowly absorbed by the body and replaced by new tissue growth, inthe shape or very nearly in the shape of the implant. The matrix alsomay be particulate in nature.

Details of how to make and how to use the matrices useful in thisinvention are disclosed below and in co-pending U.S. Ser. No. 752,764,abandoned, the disclosure of which is incorporated herein by reference.

II.B(i) Tissue-Derived Matrices

Suitable biocompatible, in vivo biodegradable acellular matrices may beprepared from naturally-occurring tissue. The tissue is treated withsuitable agents to substantially extract the cellular, nonstructuralcomponents of the tissue. The agents also should be capable ofextracting any morphogenesis inhibiting components associated with thetissue. The resulting material is a porous, acellular matrix,substantially depleted in nonstructurally-associated components.

The matrix also may be further treated with agents that modify thematrix, increasing the number of pores and micropits on its surfaces.Those skilled in the art will know how to determine which agents arebest suited to the extraction of nonstructural components for differenttissues. For example, soft tissues such as liver and lung may bethin-sectioned and exposed to a nonpolar solvent such as, for example,100% ethanol, to destroy the cellular structure of the tissue andextract nonstructural components. The material then may be dried andpulverized to yield nonadherent porous particles or it may be maintainedas a gel-like solution. Structural tissues such as cartilage and dentinwhere collagen is a primary proteinaceous component may be demineralizedand extracted with guanidinium hydrochloride, essentially following themethod of Sampath et al. (1983) PNAS 80:6591-6595. For example,pulverized and demineralized dentin is extracted with five volumes of 4Mguanidinium-HCl, 50 mM Tris-HCl, pH 7.0 for 16 hours at 4° C. Thesuspension then is filtered. The insoluble material that remains iscollected and used to fabricate the matrix. The material is mostlycollagenous in matter. It is devoid of morphogenic activity. The matrixparticles may further be treated with a collagen fibril-modifying agentthat extracts potentially unwanted components from the matrix, andalters the surface structure of the matrix material. Useful agentsinclude acids, organic solvents or heated aqueous media. A detaileddescription of these matrix treatments are disclosed, for example, inU.S. Pat. No. 4,975,526 and PCT publication U.S. No. 90/00912, publishedSep. 7, 1990 (W090/10018).

The currently most preferred agent is a heated aqueous fibril-modifyingmedium such as water, to increase the matrix particle surface area andporosity. The currently most preferred aqueous medium is an acidicaqueous medium having a pH of less than about 4.5, e.g., within therange of about pH 2-pH 4 which may help to "swell" the collagen beforeheating. 0.1% acetic acid, which has a pH of about 3, currently is mostpreferred. 0.1M acetic acid also may be used.

Various amounts of delipidated, demineralized guanidine-extracted bonecollagen are heated in the aqueous medium (1 g matrix/30 ml aqueousmedium) under constant stirring in a water jacketed glass flask, andmaintained at a given temperature for a predetermined period of time.Preferred treatment times are about one hour, although exposure times ofbetween about 0.5 to two hours appear acceptable. The temperatureemployed is held constant at a temperature within the range of about 37°C. to 65° C. The currently preferred heat treatment temperature iswithin the range of about 45° C. to 60° C.

After the heat treatment, the matrix is filtered, washed, lyophilizedand used for implant. Where an acidic aqueous medium is used, the matrixalso is preferably neutralized prior to washing and lyophilization. Acurrently preferred neutralization buffer is a 200 mM sodium phosphatebuffer, pH 7.0. To neutralize the matrix, the matrix preferably first isallowed to cool following thermal treatment, the acidic aqueous medium(e.g., 0.1% acetic acid) then is removed and replaced with theneutralization buffer and the matrix agitated for about 30 minutes. Theneutralization buffer then may be removed and the matrix washed andlyophilized.

Other useful fibril-modifying treatments include acid treatments (e.g.,trifluoroacetic acid and hydrogen fluoride) and solvent treatments suchas dichloromethane, acetonitrile, isopropanol and chloroform, as well asparticular acid/solvent combinations.

After contact with the fibril-modifying agent, the treated matrix may bewashed to remove any extracted components, following a form of theprocedure set forth below:

1. Suspend matrix preparation in TBS (Tris-buffered saline) 1 g/200 mland stir at 4° C. for 2 hrs; or in 6M urea, 50 mM Tris-HCl, 500 mM NaCl,pH 7.0 (UTBS) or water and stir at room temperature (RT) for 30 minutes(sufficient time to neutralize the pH);

2. Centrifuge and repeat wash step; and

3. Centrifuge; discard supernatant; water wash residue; and thenlyophilize.

Alternatively, suitable matrix materials may be obtained commercially.For example, an extracellular matrix extract such as Matrigel™,(Collaborative Research, Inc., Bedford) derived from mouse sarcomacells, may be used to advantage.

II.B(ii) Synthetic Matrices

In addition to the naturally-derived tissue-specific matrices describedabove, useful tissue-specific matrices may be formulated synthetically.These porous biocompatible, in vivo biodegradable synthetic matrices aredisclosed in PCT publication U.S. 91/03603, published Dec. 12, 1991(W091/18558), the disclosure of which is hereby incorporated byreference. Briefly, the matrix comprises a porous crosslinked structuralpolymer of biocompatible, biodegradable collagen and appropriate,tissue-specific glycosaminoglycans as tissue-specific cell attachmentfactors. Collagen derived from a number of sources may be suitable foruse in these synthetic matrices, including insoluble collagen,acid-soluble collagen, collagen soluble in neutral or basic aqueoussolutions, as well as those collagens which are commercially available.

Glycosaminoglycans (GAGs) or mucopolysaccharides arehexosamine-containing polysaccharides of animal origin that have atissue specific distribution, and therefore may be used to helpdetermine the tissue specificity of the morphogen-stimulateddifferentiating cells. Reaction with the GAGs also provides collagenwith another valuable property, i.e., inability to provoke an immunereaction (foreign body reaction) from an animal host.

Chemically, GAGs are made up of residues of hexosamines glycosidicallybound and alternating in a more-or-less regular manner with eitherhexouronic acid or hexose moieties (see, e.g., Dodgson et al. inCarbohydrate Metabolism and its Disorders (Dickens et al., eds.) Vol. 1,Academic Press (1968)). Useful GAGs include hyaluronic acid, heparin,heparin sulfate, chondroitin 6-sulfate, chondroitin 4-sulfate, dermatansulfate, and keratin sulfate. Other GAGs are suitable for forming thematrix described herein, and those skilled in the art will either knowor be able to ascertain other suitable GAGs using no more than routineexperimentation. For a more detailed description of mucopolysaccharides,see Aspinall, Polysaccharides, Pergamon Press, Oxford (1970). Forexample, as disclosed in U.S. application Ser. No. 529,852, abandoned,chondroitin-6-sulfate can be used where endochondral bone formation isdesired. Heparin sulfate, on the other hand, may be used to formulatesynthetic matrices for use in lung tissue repair.

Collagen can be reacted with a GAG in aqueous acidic solutions,preferably in diluted acetic acid solutions. By adding the GAG dropwiseinto the aqueous collagen dispersion, coprecipitates of tangled collagenfibrils coated with GAG results. This tangled mass of fibers then can behomogenized to form a homogeneous dispersion of fine fibers and thenfiltered and dried.

Insolubility of the collagen-GAG products can be raised to the desireddegree by covalently cross-linking these materials, which also serves toraise the resistance to resorption of these materials. In general, anycovalent cross-linking method suitable for cross-linking collagen alsois suitable for cross-linking these composite materials, althoughcrosslinking by a dehydrothermal process is preferred.

When dry, the crosslinked particles are essentially spherical, withdiameters of about 500 μm. Scanning electron miscroscopy shows pores ofabout 20 μm on the surface and 40 μm on the interior. The interior ismade up of both fibrous and sheet-like structures, providing surfacesfor cell attachment. The voids interconnect, providing access to thecells throughout the interior of the particle. The material appears tobe roughly 99.5% void volume, making the material very efficient interms of the potential cell mass that can be grown per gram ofmicrocarrier.

Another useful synthetic matrix is one formulated from biocompatible, invivo biodegradable synthetic polymers, such as those composed ofglycolic acid, lactic acid and/or butyric acid, including copolymers andderivatives thereof. These polymers are well described in the art andare available commercially. For example, polymers composed of polyacticacid (e.g., MW 100 kDa), 80% polylactide/20% glycoside or poly3-hydroxybutyric acid (e.g., MW 30 kDa) all may be purchased fromPolySciences, Inc. The polymer compositions generally are obtained inparticulate form. In addition, one can alter the morphology of thepolymer compositions, for example to increase porosity, using any of anumber of particular solvent treatments known in the art. Where themorphogen is adsorbed to the matrix surface, the steps preferably areperformed under conditions which avoid hydrolysis of the polymers (e.g.,non-aqueous conditions such as in an ethanol-trifluoro-acetic acidsolution).

The OP-3 proteins described herein can be combined and dispersed in asuitable matrix using any of the methods described below:

1. Ethanol Precipitation

Matrix is added to the morphogen dissolved in guanidine-HCl. Samples arevortexed and incubated at a low temperature. Samples are then furthervortexed. Cold absolute ethanol is added to the mixture which is thenstirred and incubated. After centrifugation (microfuge, high speed) thesupernatant is discarded. The matrix is washed with cold concentratedethanol in water and then lyophilized.

2. Acetonitrile Trifluoroacetic Acid Lyophilization

In this procedure, a morphogenically active fragment of OP-3 in anacetonitrile trifluroacetic acid (ACN/TFA) solution is added to thecarrier material. Samples are vigorously vortexed many times and thenlyophilized.

3. Buffered Saline Lyophilization

A preparation of a morphogenically active fragment of OP-3 inphysiological saline also may be vortexed with the matrix andlyophilized to produce morphogenically active material.

Tissue morphogenesis requires a morphogenically permissive environment.Clearly, in fully-functioning healthy tissue that is not composed of apermanently renewing cell population, there must exist signals toprevent continued tissue growth. Thus, it is postulated that thereexists a control mechanism, such as a feedback control mechanism, whichregulates the control of cell growth and differentiation. In fact, it isknown that both TGF-β, and MIS are capable of inhibiting cell growthwhen present at appropriate concentrations. In addition, using the bonemodel system it can be shown that osteogenic devices comprising abone-derived carrier (matrix) that has been demineralized andguanidine-extracted to substantially remove the noncollagenous proteinsdoes allow endochondral bone formation when implanted in associationwith an osteoinductive morphogen. If, however, the bone-derived carrieris not demineralized but rather is washed only in low salt, for example,induction of endochondral bone formation is inhibited, suggesting thepresence of one or more inhibiting factors within the carrier.

III. EXAMPLES

Example 1 Recombinant Production of OP-3

OP-3 proteins useful in the methods and compositions of this inventionmay be purified from natural sources or produced using standardrecombinant methodology. General considerations for the recombinantproduction of morphogens are described in U.S. Ser. No. 752,764,abandoned, the disclosure of which is incorporated hereinabove byreference.

A. Identification of Novel mOP-3 Sequences

A genetic sequence encoding the morphogenic OP-3 protein was identifiedusing a 0.3 kb EcoRI-BamH1 OP-2 fragment from a mouse OP-2 cDNA as ahybridization probe, specific to the mid-pro region of OP-2(corresponding to amino acid residues 125 to 225 of the pre-pro protein)essentially as described in U.S. Ser. No. 667,274, abandoned. The ³²P-labeled probe was prepared using the random hexanucleotide primingmethod, and the hybridizations were performed using the followingconditions: 40% formamide, 5× SSPE, 5× Denhardt's Solution, 0.1% SDS, at37° C. overnight, and washing in 0.1× SSPE, 0.1% SDS at 50° C.Approximately 1×10⁶ phages from a mouse cDNA (carried in lambda zapII)library made from the teratocarcinoma cell line PCC4 (Stratagene, Inc.,La Jolla, Calif., cat #936301) were screened. This screening yieldedfour individual clones which were purified over three rounds ofscreening. The plasmid DNA containing the cDNAs was obtained using thelambda zapII excision process following manufacturer's directions. Threeof the four clones were shown by DNA sequencing to encode OP-3. The DNAsequence, referred to herein as mOP-3 and described in Seq. ID No. 1,was identified by this procedure.

The isolated mOP-3 DNA sequence, in accordance with other knownmorphogens, encodes a protein comprising a "pro" region (definedessentially by residues 20-260 or 20-263 of Seq. ID No. 1) and a matureregion (defined essentially by residues 261-399 or 264-399 of Seq. IDNo. 1), including a functional domain comprising the conserved cysteineskeleton.

Like OP-2, OP-3 is marked by an eighth cysteine within the sevencysteine domain (e.g., at position 338 of Seq. ID No.1). The extracysteine likely helps stabilize the folded structure, possibly byproviding inter-molecular disulfide bonding. The extra cysteine alsoallows for heterodimer formation between OP-3 and another morphogencomprising the "eighth" cysteine, like OP-2 for example, or a modifiedOP-1, wherein an extra cysteine has been inserted at the appropriatelocation. The extra cysteine also may allow tetramer formation. Theextra cysteine does not inhibit synthesis or reduce the stability of thetranslated sequence significantly as expressed proteins comprising theextra cysteine are readily detected by SDS gel electrophoresis. Aprimary glycosylation site occurs just C terminal to the extra cysteinein both OP-2 and OP-3, which may provide a protective effect.

The cDNA sequences for both human and mouse OP-2 are provided in Seq. IDNos. 7 and 9, and the genomic sequence for human OP-2 is provided inSeq. ID No. 11, wherein the exons defining the coding region of theseproteins are indicated. The exon boundaries also are indicated in FIG.1, described below. The human OP-2 locus was isolated from a genomiclibrary (Clontech, EMBL-3 #HL1067J) on three overlapping phage clones,using standard cloning procedures. The OP-2 coding information wasspread over 27 kb and, like OP-1, contains 7 exons. A comparison ofexon-intron boundaries in the 7 cysteine domain showed matchinglocations with those of OP-1. The first OP-2 exon contains 334 bp ofcoding sequence (111 amino acids), including the signal peptide, and isfollowed by the largest intron (14.6 kb). The second exon (190 bp, 64amino acids) is separated by a short intron (0.4 kb) from exon 3 (149bp, 49 amino acids). It follows a large third intron of 9.5 kb. Thefourth exon (195 bp, 65 amino acids) encodes the maturation site("OP-2-Ala") and is followed by a 0.8 kb intron. The 7 cysteine domainsresides on exons 5 to 7: exon 5 (80 bp, 27 amino acids) encodes thefirst cysteine of mature OP-2 and is followed by intron 5 (0.5 kb inlength), exon 6 (111 bp, 37 amino acids) is separated by a 2.5 kb intronfrom the seventh, last exon with 147 bp (49 amino acids) of codingsequence. As stated above, the exon-intron boundaries are conservedbetween human OP-1 and OP-2, two different members of the morphogenfamily of proteins. By analogy, the exon-intron boundaries between humanand mouse OP-2, two species variants of a morphogen, are anticipated tobe conserved as well.

FIG. 1 shows the alignment of the murine OP-2 and murine OP-3 codingregions of the cDNA. The exon boundaries are indicated by bars beneaththe sequence. Both sequences have the same number of nucleotides. Thenucleotide sequence is about 80% conserved in the N-terminal andC-terminal regions. In the figure, nucleotide identity between thesequences is indicated by stippling. In addition, the central region ofthe sequence is highly conserved and this conserved region falls intothe boundaries of exon 2 and 3. There are only three nucleotidedifferences in this region, indicated in the figure by diamonds.

The high degree of conservation in the nucleotide sequences indicatesthat OP-2 and OP-3 likely share the nucleotide sequence of exon 2 and 3.The different proteins may result from alternatively splicedtranscripts, or they may arise from independent genes which share partof their coding sequence. Intron 1, which lies upstream of exon 2 inOP-2 (see Seq. ID No. 11) is large (14.6 kb) and could include the startof the OP-3 gene and/or its first exon sequence. Certainly, as has beenfound for other mammalian genes, one or more of the introns of thesemorphogens may include sequences having a transcription regulatoryfunction.

Using the screening procedure described herein and in U.S. Ser. No.752,764, abandoned, and the labelled OP-2 fragment, or preferably alabelled OP-3 fragment, OP-3 genetic sequences from other species andother libraries may be isolated. Alternatively, or in addition, a probeto the N-terminal region of the mature protein, or the 3' noncodingregion flanking and immediately following the stop codon, also may beused to screen for other OP-3 species variants. These sequences varysubstantially among the morphogens and represent morphogen-specificsequences. Mammalian cell expression of OP-3 readily can be achievedusing COS (simian kidney ATCC, CRL-1650) or

CHO (Chinese hamster ovary) cells (e.g., CHO-DXBII, from LawrenceChasin, Columbia University, New York). An exemplary protocol formammalian cell expression is provided below. Other useful eukaryoticcell systems include the insect/baculovirus system or the mammaliancomplement system.

B. Expression of Novel OP-3 Sequences

To express the OP=3 protein, the OP-3 DNA is subcloned into an insertionsite of a suitable, commercially available pUC-type vector (e.g.,pUC-19, ATCC #37254, Rockville, Md.), along with a suitablepromoter/enhancer sequences and 3' termination sequences. Currentlypreferred promoter/enhancer sequences are the CMV promoter (humancytomegalovirus major intermediate--early promoter) and the mousemammary tumor virus promoter (mMTV) boosted by the rous sarcoma virusLTR enhancer sequence (e.g., from Clontech, Inc., Palo Alto). Expressionalso may be further enhanced using transactivating enhancer sequences.The plasmid also contains DHFR as an amplifiable marker, under SV40early promoter control (ATCC #37148). Transfection, cell culturing, geneamplification and protein expression conditions are standard conditions,well known in the art, such as are described, for example in Ausubel etal., ed., Current Protocols in Molecular Biology, John Wiley & Sons, NewYork (1989). Briefly, transfected cells are cultured in mediumcontaining 0.1-0.5% dialyzed fetal calf serum (FCS), stably transfectedhigh expression cell lines obtained by subcloning and evaluated bystandard Northern blot. Southern blots also are used to assess the stateof integrated OP-3 sequences and the extent of their copy numberamplification.

Chimeric OP-3 morphogens, e.g., comprising an OP-3 active domain and,for example, part or all of a pro domain from another, differentmorphogen may be constructed using standard recombinant DNA technologyand/or an automated DNA synthesizer to construct the desired sequence.Useful chimeras include those wherein the non-OP-3 sequence is joined tothe OP-3 sequence encoding the mature OP-3 protein, and the non-OP-3sequence encodes part or all of the sequence between the signal peptideprocessing site and the "Arg-Xaa-Xaa-Arg" processing sequence from atleast one morphogen. Alternatively, the non-OP-3 sequence may be joinedto an OP-3 sequence encoding, for example, the 6 or 7 cysteineskeletons, wherein the non-OP-3 sequence includes the sequence encodingthe N-terminus of the mature protein. As will be appreciated by personsskilled in the art, the non-OP-3 sequences may be composed of sequencesfrom one or morphogens and/or may comprise novel biosynthetic sequences.

The expressed protein then is purified as follows. For a typical 2Lpreparation of transfected mammalian cells conditioned in 0.5% FCS, forexample, the total protein is typically about 700 mg. The amount of OP-3in the media, estimated by Western blot, is between about 0.1-5.0 mg.OP-3 media then is diluted in a low salt, physiologically buffered 6Murea solution, and loaded onto an S-Sepharose column, which acts as astrong cation exchanger. OP-3 binds to the column in low salt, and serumproteins are removed. The column subsequently is developed with an NaClgradient, e.g., 0.1M NaCl-1.0M NaCl, in 6M urea, 20 mM HEPES, pH 7.0.Most contaminants are removed at the start of the gradient, and OP-3 iseluted primarily at a higher salt concentration.

The sample then is loaded onto a phenyl-Sepharose column (hydrophobicinteraction chromatography). OP-3 binds phenyl-Sepharose in the presenceof high concentrations of a weak chaotropic salt (e.g., 1M (NH₄)₂ SO₄ ina physiologically buffered 6M urea solution). Once OP-3 is bound, thecolumn is developed with a decreasing ammonium sulfate gradient, e.g.,0.6M-0.0M (NH₄)₂ SO₄ gradient in a physiologically buffered, 6M ureasolution. Again, most contaminants are removed at the start of thegradient, and OP-3 elutes primarily at low or no ammonium sulfateconcentrations.

The OP-3 eluted from the phenyl-Sepharose column then is dialyzedagainst water, and prepared for loading onto a reverse phasechromatography column (e.g., C-18 HPLC), for example, by dialyzingagainst 30% acetonitrile, 0.1% TFA.

An alternative chromatography protocol is to perform the S-Sepharosechromatography in the absence of 6 M urea. The bound proteins then areeluted with salt step elutions (e.g., 0.1-0.6M NaCl). Remaining OP-3then can be eluted in the presence of 6M urea. The 6M urea elution alsomay be used in place of the non-urea elution to achieve maximum recoveryin one step. In addition, OP-3 may be eluted from the phenyl-Sepharosecolumn in 38% ethanol-0.01% TFA, thereby eliminating the need to dialyzethe eluent before applying it to the C-18 column. Finally, multiple C-18columns may be used (e.g., three), to further enhance purification andconcentration of the protein.

OP-3 also will bind hydroxyapatite efficiently, typically in the absenceof 6M urea and at low phosphate concentrations (less than 5 mMphosphate). Bound OP-3 can be removed from the column with an elutiongradient of about 0.001-0.5M step elution of phosphate in aphysiologically buffered solution. Additionally, urea (6M) may be addedduring the elution step.

Other related chromatography methods also may be useful in purifyingOP-3 from eucaryotic cell culture systems. For example,heparin-Sepharose may be used in combination with the S-Sepharosecolumn. Alternatively, immobilized metal-ion affinity chromatography(IMAC) (e.g., Cu²⁺ or Zn⁺) and a physiologically buffered phosphatesolution may be used to advantage.

Example 2 Mitogenic Effect of OP-3

2.1 Mitogenic Effect of Morphogen on Rat and Human Osteoblasts

The following example demonstrates the ability of OP-3 to induceproliferation of osteoblasts in vitro using the following assay. In thisand all examples involving osteoblast cultures, rat osteoblast-enrichedprimary cultures preferably are used. Although these cultures areheterogeneous in that the individual cells are at different stages ofdifferentiation, the culture is believed to more accurately reflect themetabolism and function of osteoblasts in vivo than osteoblast culturesobtained from established cell lines. Unless otherwise indicated, allchemicals referenced are standard, commercially available reagents,readily available from a number of sources, including Sigma Chemical,Co., St. Louis; Calbiochem, Corp., San Diego and Aldrich Chemical Co.,Milwaukee.

Rat osteoblast-enriched primary cultures are prepared by sequentialcollagenase digestion of newborn suture-free rat calvaria (e.g., from1-2 day-old animals, Long-Evans strain, Charles River Laboratories,Wilmington, Mass.), following standard procedures, such as aredescribed, for example, in Wong et al., (1975) PNAS 72:3167-3171. Ratosteoblast single cell suspensions then are plated onto a multi-wellplate (e.g., a 24 well plate) at a concentration of 50,000 osteoblastsper well in alpha MEM (modified Eagle's medium, Gibco, Inc., LongIsland) containing 10% FBS (fetal bovine serum), L-glutamine andpenicillin/streptomycin. The cells are incubated for 24 hours at 37° C.,at which time the growth medium is replaced with alpha MEM containing 1%FBS and the cells incubated for an additional 24 hours so that cells arein serum-deprived growth medium at the time of the experiment.

The cultured cells are divided into three groups: (1) wells whichreceive, for example, 0.1, 1.0, 10.0, 40 and 80.0 ng of OP-3; (2) wellswhich receive 0.1, 1.0, 10.0 and 40 ng of a local-acting growth factor(e.g., TGF-β); and (3) the control group, which receive no growthfactors. The cells then are incubated for an additional 18 hours afterwhich the wells are pulsed with 2 μCi/well of ³ H-thymidine andincubated for six more hours. The excess label then is washed off with acold solution of 0.15M NaCl and then 250 μl of 10% tricholoracetic acidis added to each well and the wells incubated at room temperature for 30minutes. The cells then are washed three times with cold distilledwater, and lysed by the addition of 250 μl of 1% sodium dodecyl sulfate(SDS) for a period of 30 minutes at 37° C. The resulting cell lysatesare harvested using standard means well known in the art, and theincorporation of ³ H-thymidine into cellular DNA determined by liquidscintillation as an indication of mitogenic activity of the cells. Inthe experiment, OP-3 stimulates ³ H-thymidine incorporation into DNA,and thus promote osteoblast cell proliferation. By contrast, the effectof TGF-β is transient and biphasic. At high concentrations, TGF-β has nosignificant effect on osteoblast cell proliferation.

The in vitro effect of OP-3 on osteoblast proliferation also may beevaluated using human primary osteoblasts (obtained from bone tissue ofa normal adult patient and prepared as described above) and on humanosteosarcoma-derived cell lines. In all cases OP-3 induces cellproliferation in accordance with the morphogen's ability to induceendochondral bone formation (see Example 7, below).

2.2 Progenitor Cell Stimulation

The following example demonstrates the ability of OP-3 to stimulate theproliferation of mesenchymal progenitor cells. Useful naive stem cellsinclude pluripotential stem cells, which may be isolated from bonemarrow or umbilical cord blood using conventional methodologies, (see,for example, Faradji et al., (1988) Vox Sang., 55 (3):133-138 orBroxmeyer et al., (1989) PNAS 86:3828-3832), as well as naive stem cellsobtained from blood. Alternatively, embryonic cells (e.g., from acultured mesodermal cell line) may be useful.

Another method for obtaining progenitor cells and for determining theability of OP-3 fragments to stimulate cell proliferation is to captureprogenitor cells from an in vivo source. For example, a biocompatiblematrix material able to allow the influx of migratory progenitor cellsmay be implanted at an in vivo site long enough to allow the influx ofmigratory progenitor cells. For example, a bone-derived,guanidine-extracted matrix, formulated as disclosed for example inSampath et al. ((1983) PNAS 80:6591-6595), or U.S. Pat. No. 4,975,526,may be implanted into a rat at a subcutaneous site, essentiallyfollowing the method of Sampath et al. After three days the implant isremoved, and the progenitor cells associated with the matrix dispersedand cultured.

Progenitor cells, however obtained, then are incubated in vitro withOP-3 under standard cell culture conditions well described in the artand described hereinabove. In the absence of external stimuli, theprogenitor cells do not, or only minimally, proliferate on their own inculture. However, progenitor cells cultured in the presence of amorphogenically active fragment of OP-3 do proliferate. Cell growth canbe determined visually or spectrophotometrically using standard methodswell known in the art.

Example 3 Morphogen-Induced Cell Differentiation

3.1 Embryonic Mesenchyme Differentiation

Morphogenically active fragments of OP-3 can be utilized to induce celldifferentiation. The ability of OP-3 to induce cell differentiation canbe demonstrated by culturing early mesenchymal cells in the presence ofOP-3 and then studying the histology of the cultured cells by stainingwith toluidine blue using standard cell culturing and cell stainingmethodologies well described in the art. For example, it is known thatrat mesenchymal cells destined to become mandibular bone, when separatedfrom the overlying epithelial cells at stage 11 and cultured in vitrounder standard tissue culture conditions, e.g., in a chemically defined,serum-free medium, containing for example, 67% DMEM (Dulbecco's modifiedEagle's medium), 22% F-12 medium, 10 mM Hepes pH 7, 2 mM glutamine, 50μg/ml transferrin, 25 μg/ml insulin, trace elements, 2 mg/ml bovineserum albumin coupled to oleic acid, with HAT (0.1 mM hypoxanthine, 10μM aminopterin, 12 μM thymidine, will not continue to differentiate.However, if these same cells are left in contact with the overlyingendoderm for an additional day, at which time they become stage 12cells, they will continue to differentiate on their own in vitro to formchondrocytes. Further differentiation into osteoblasts and, ultimately,mandibular bone, requires an appropriate local environment, e.g., avascularized environment.

Stage 11 mesenchymal cells, cultured in vitro in the presence of OP-3,e.g., 10-100 ng/ml, will continue to differentiate in vitro to formchondrocytes just as they continue to differentiate in vitro if they arecultured with the cell products harvested from the overlying endodermalcells. This experiment may be performed with different mesenchymal cellsto demonstrate the cell differentiation capability of OP-3 in differenttissues.

As another example of morphogen-induced cell differentiation, theability of OP-3 to induce osteoblast differentiation may be demonstratedin vitro using primary osteoblast cultures, or osteoblast-like cellslines, and assaying for a variety of bone cell markers that are specificmarkers for the differentiated osteoblast phenotype, e.g., alkalinephosphatase activity, parathyroid hormone-mediated cyclic AMP (cAMP)production, osteocalcin synthesis, and enhanced mineralization rates.

3.2 Alkaline Phosphatase Induction of Osteoblasts by OP-3

The cultured cells in serum-free medium are incubated with, a range ofOP-3 concentrations, for example, 0.1, 1.0, 10.0, 40.0 or 80.0 ngOP-3/ml medium; or with a similar range of TGF-β concentrations. 72hours after the incubation period the cell layer is extracted with 0.5ml of 1% Triton X-100. The resultant cell extract then, is centrifuged,and 100 μl of the extract is added to 90 μl of paranitrosophenylphospate(PNPP)/glycerine mixture and incubated for 30 minutes in a 37° C. waterbath and the reaction stopped with 100 μl NaOH. The samples then are runthrough a plate reader (e.g., Dynatech MR700 plate reader, andabsorbance measured at 400 nm, using p-nitrophenol as a standard) todetermine the presence and amount of alkaline phosphate activity.Protein concentrations are determined by the Biorad method. Alkalinephosphatase activity is calculated in units/μg protein, where 1 unit=1nmol p-nitrophenol liberated/30 minutes at 37° C.

OP-3 alone stimulates the production of alkaline phosphatase inosteoblasts, and thus promotes the growth and expression of theosteoblast differentiated phenotype.

The long term effect of OP-3 morphogen on the production of alkalinephosphatase by rat osteoblasts also may be demonstrated as follows.

Rat osteoblasts are prepared and cultured in multi-well plates asdescribed above. In this example six sets of 24 well plates are platedwith 50,000 rat osteoblasts per well. The wells in each plate, preparedas described above, then are divided into three groups: (1) those whichreceive, for example, 1 ng of OP-3 per ml of medium; (2) those whichreceive 40 ng of OP-3 per ml of medium; and (3) those which received 80ng of OP-3 per ml of medium. Each plate then is incubated for differentlengths of time: 0 hours (control time), 24 hours, 48 hours, 96 hours,120 hours and 144 hours. After each incubation period, the cell layer isextracted with 0.5 ml of 1% Triton X-100 . The resultant cell extract iscentrifuged, and alkaline phosphatase activity determined as for Example3.1, using paranitroso-phenylphosphate (PNPP). OP-3 stimulates theproduction of alkaline phosphatase in osteoblasts in dose-dependentmanner so that increasing doses of OP-3 further increase the level ofalkaline phosphatase production, and moreover, the OP-3-stimulatedelevated levels of alkaline phosphatase in the treated osteoblasts isanticipated to last for an extended period of time.

3.3 OP-3 Protein Induction of PTH-Mediated cAMP.

The effect of a OP-3 on parathyroid hormone-mediated cAMP production inrat osteoblasts in vitro may be demonstrated as follows.

Rat osteoblasts are prepared and cultured in a multiwell plate asdescribed above. The cultured cells then are divided into three groups:(1) wells which receive, for example, 1.0, 10.0 and 40.0 ng OP-3/mlmedium); (2) wells which receive for example, TGF-β, at similarconcentration ranges; and (3) a control group which receives no growthfactors. The plate is then incubated for another 72 hours. At the end ofthe 72 hours the cells are treated with medium containing 0.5% bovineserum albumin (BSA) and 1 mM 3-isobutyl-1-methylxanthine for 20 minutesfollowed by the addition into half of the wells of human recombinantparathyroid hormone (hPTH, Sigma, St. Louis) at a concentration of 200ng/ml for 10 minutes. The cell layer then is extracted from each wellwith 0.5 ml of 1% Triton X-100. The cAMP levels then are determinedusing a radioimmunoassay kit (e.g., Amersham, Arlington Heights, Ill.).OP-3 alone stimulates an increase in the PTH-mediated cAMP response, andthus promotes the growth and expression of the osteoblast differentiatedphenotype.

3.4 OP-3 Protein Induction of Osteocalcin Production

Osteocalcin is a bone-specific protein synthesized by osteoblasts whichplays an integral role in the rate of bone mineralization in vivo.Circulating levels of osteocalcin in serum are used as a marker forosteoblast activity and bone formation in vivo. Induction of osteocalcinsynthesis in osteoblast-enriched cultures can be used to demonstrateOP-3 morphogenic efficacy in vitro.

Rat osteoblasts are prepared and cultured in a multi-well plate asabove. In this experiment the medium is supplemented with 10%FBS, and onday 2, cells are fed with fresh medium supplemented with fresh 10 mMβ-glycerophosphate (Sigma, Inc.). Beginning on day 5 and twice weeklythereafter, cells are fed with a complete mineralization mediumcontaining all of the above components plus fresh L(+)-ascorbate, at afinal concentration of 50 μg/ml medium. OP-3 then is added to the wellsdirectly, e.g., in 50% acetonitrile (or 50% ethanol) containing 0.1%trifluoroacetic acid (TFA), at no more than 5 μl morphogen/ml medium.Control wells receive solvent vehicle only. The cells then are re-fedand the conditioned medium sample diluted 1:1 in standardradioimmunoassay buffer containing standard protease inhibitors andstored at -20° C. until assayed for osteocalcin. Osteocalcin synthesisis measured by standard radioimmunoassay using a commercially availableosteocalcin-specific antibody.

Mineralization is determined on long term cultures (13 day) using amodified yon Kossa staining technique on fixed cell layers: cells arefixed in fresh 4% paraformaldehyde at 23° C. for 10 min, followingrinsing cold 0.9% NaCl. Fixed cells then are stained for endogenousalkaline phosphatase at pH 9.5 for 10 min, using a commerciallyavailable kit (Sigma, Inc.) Purple stained cells then are dehydratedwith methanol and air dried. after 30 min incubation in 3% AgNO₃ in thedark, H₂ O-rinsed samples are exposed for 30 sec to 254 nm UV light todevelop the black silver-stained phosphate nodules. Individualmineralized foci (at least 20 μm in size) are counted under a dissectingmicroscope and expressed as nodules/culture.

OP-3 stimulates osteocalcin synthesis in osteoblast cultures. Theincreased osteocalcin synthesis in response to OP-3 is dose dependentand shows a significant increase over the basal level after 13 days ofincubation. The enhanced osteocalcin synthesis also can be confirmed bydetecting the elevated osteocalcin mRNA message (20-fold increase) usinga rat osteocalcin-specific probe. In addition, the increase inosteoclacin synthesis correlates with increased mineralization in longterm osteoblast cultures as determined by the appearance of mineralnodules. OP-3 increases the initial mineralization rate significantlycompared to untreated cultures.

3.5 Morphogen-Induced CAM Expression

The morphogens described herein induce CAM expression, particularlyN-CAM expression, as part of their induction of morphogenesis (seecopending U.S. Ser. No. 922,813, abandoned). CAMs are morphoregulatorymolecules identified in all tissues as an essential step in tissuedevelopment. N-CAMs, which comprise at least 3 isoforms (N-CAM-180,N-CAM-140 and N-CAM-120, where "180", "140" and "120" indicate theapparent molecular weights of the isoforms as measured by SDSpolyacrylamide gel electrophoresis) are expressed at least transientlyin developing tissues, and permanently in nerve tissue. Both theN-CAM-180 and N-CAM-140 isoforms are expressed in both developing andadult tissue. The N-CAM-120 isoform is found only in adult tissue.Another neural CAM is L1.

The ability of OP-3 to stimulate CAM expression can be demonstratedusing the following protocol, using NG108-15 cells. NG108-15 is atransformed hybrid cell line (neuroblastoma×glioma, America Type TissueCulture (ATCC), Rockville, Md.), exhibiting a morphology characteristicof transformed embryonic neurons. As described in Example 4, below,untreated NG108-15 cells exhibit a fibroblastic, or minimallydifferentiated, morphology and express only the 180 and 140 isoforms ofN-CAM normally associated with a developing cell. Following morphogentreatment these cells exhibit a morphology characteristic of adultneurons and express enhanced levels of all three N-CAM isoforms.

In this example, NG108-15 cells are cultured for 4 days in the presenceof increasing concentrations of OP-3 using standard culturingprocedures, and standard Western blots performed on whole cell extracts.N-CAM isoforms are detected with an antibody which crossreacts with allthree isoforms, mAb H28.123, obtained from Sigma Chemical Co., St.Louis, the different isoforms being distinguishable by their differentmobilities on an electrophoresis gel. Control NG108-15 cells (untreated)express both the 140 kDa and the 180 kDa isoforms, but not the 120 kDa,as determined by Western blot analyses using up to 100 μg of protein.Treatment of NG108-15 cells with OP-3 results in a dose-dependentincrease in the expression of the 180 kDa and 140 kDa isoforms, as wellas the induction of the 120 kDa isoform induced. In addition,OP-3-induced CAM expression correlates with cell aggregation, asdetermined by histology.

Example 4 OP-3 Protein-Induced Redifferentiation of TransformedPhenotype

The OP-3 morphogens described herein also can induce redifferentiationof transformed cells to a morphology characteristic of untransformedcells. The examples provided below detail morphogen-inducedredifferentiation of a transformed human cell line of neuronal origin(NG108-15); as well as mouse neuroblastoma cells (N1E-115), and humanembryo carcinoma cells, to a morphology characteristic of untransformedcells.

As described above, NG108-15 is a transformed hybrid cell line producedby fusing neuroblastoma×glioma cells (obtained from ATTC, Rockville,Md.), and exhibiting a morphology characteristic of transformedembryonic neurons, e.g., having a fibroblastic morphology. Specifically,the cells have polygonal cell bodies, short, spike-like processes andmake few contacts with neighboring cells (see copending U.S. Ser. No.922,813, abandoned). Incubation of NG108-15 cells, cultured in achemically defined, serum-free medium, with 0.1 to 300 ng/ml ofmorphogen (e.g; OP-3) for four hours induces an orderly, dose-dependentchange in cell morphology.

In the example, NG108-15 cells are subcultured on poly-L-lysine coated 6well plates. Each well contains 40-50,000 cells in 2.5 ml of chemicallydefined medium. On the third day, 2.5 μl of morphogen (e.g., OP-3) in60% ethanol containing 0.025% trifluoroacetic is added to each well.Morphogenic OP-3 of varying concentrations are tested (typically,concentration ranges of 0-300 ng/ml are tested). The media is changeddaily with new aliquots of morphogen. OP-3 induces a dose-dependentredifferentiation of the transformed cells, including a rounding of thesoma, an increase in phase brightness, extension of the short neuriteprocesses, and other significant changes in the cellular ultrastructure.After several days treated cells begin to form epithelioid sheets thatthen become highly packed, multi-layered aggregates, as determinedvisually by microscopic examination.

Moreover, morphogen-induced redifferentiation occurs without anyassociated changes in DNA synthesis, cell division, or cell viability,making it unlikely that the morphologic changes are secondary to celldifferentiation or a toxic effect of the morphogen. In addition, themorphogen-induced redifferentiation does not inhibit cell division, asdetermined by ³ H-thymidine uptake, unlike other molecules which havebeen shown to stimulate differentiation of transformed cells, such asbutyrate, DMSO, retanoic acid or Forskolin in analogous experiments.Thus, OP-3 maintains cell stability and viability after inducingredifferentiation.

The OP-3 morphogens described herein accordingly provide usefultherapeutic agents for the treatment of neoplasias and neoplasticlesions of the nervous system, particularly in the treatment ofneuroblastomas, including retinoblastomas, and gliomas.

As yet another, related example, the ability of OP-3 to induce the"redifferentiation" of transformed human cells may be demonstrated usingthe following assay. Specifically, the effect of OP-3 on human EC cells(embryo carcinoma cells, e.g., NTERA-Z CL.D1, ATCC, Rockville, Md.) maybe determined. In the absence of an external stimulant, these cells canbe maintained as undifferentiated stem cells, and can be induced to growin serum free media (SFM). In the absence of treatment with a morphogen,the cells proliferate rampantly and are anchorage-independent. In thepresence of morphogen, EC cells grow as flattened cells, becominganchorage dependent and forming aggregates. In addition, growth rate isreduced approximately 10 fold. Ultimately, the cells are induced todifferentiate. In the example, varying concentrations of OP-3 (e.g.,0-300 ng/ml) are added daily to cultured cells (e.g., 40-50,000 cells in2.5 ml chemically defined medium), and the effects of treatmentdetermined by visual examination. OP-3 stimulates redifferentiation ofthese cells to a morphology characteristic of untransformed embryocells.

Example 5 Maintenance of Phenotype

Morphogenically active fragments of OP-3 also may be used to maintain acell's differentiated phenotype. This application is particularly usefulfor inducing the continued expression of phenotype in senescent orquiescent cells.

5.1 In Vitro Model for Phenotypic Maintenance

The phenotypic maintenance capability of morphogens is determinedreadily. A number of differentiated cells become senescent or quiescentafter multiple passages in vitro under standard tissue cultureconditions well described in the art (e.g., Culture of Animal Cells: AManual of Basic Techniques, C. R. Freshney, ed., Wiley, 1987). However,if these cells are cultivated in vitro in association with a morphogensuch as OP-3, cells are stimulated to maintain expression of theirphenotype through multiple passages. For example, the alkalinephosphatase activity of cultured osteoblasts, such as culturedosteosarcoma cells and calvaria cells, is significantly reduced aftermultiple passages in vitro. However, if the cells are cultivated in thepresence of OP-3, alkaline phosphatase activity is maintained overextended periods of time. Similarly, phenotypic expression of myocytesalso is maintained in the presence of a morphogen. In the experiment,osteoblasts are cultured as described in Example 2. The cells aredivided into groups, incubated with varying concentrations of OP-3(e.g., 0-300 ng/ml) and passaged multiple times (e.g., 3-5 times) usingstandard methodology. Passaged cells then are tested for alkalinephosphatase activity, as described in Example 3 as an indication ofdifferentiated cell metabolic function. Osteoblasts cultured in theabsence of OP-3 have reduced alkaline phosphatase activity, as comparedto OP-3-treated cells.

5.2 In Vivo Model for Phenotypic Maintenance

Phenotypic maintenance capability also may be demonstrated in vivo,using a rat model for osteoporosis, as disclosed in the copending U.S.Ser. No. 752,857, filed Aug. 30, 1991, abandoned, and U.S. Ser. No.923,780, abandoned, incorporated hereinabove. As described therein, LongEvans female rats (Charles River Laboratories, Wilmington, Mass.) areSham-operated (control animals) or ovariectomized using standardsurgical techniques, to produce an osteoporotic condition resulting fromdecreased estrogen production. Shortly following surgery, e.g., 200 daysafter ovariectomy, rats are systemically provided with phosphatebuffered saline (PBS) or morphogen, (e.g., OP-3, 1-100 μg) for 21 days(e.g., by daily tail vein injection. ) The rats then are sacrificed andserum alkaline phosphatase levels, serum calcium levels, and serumosteocalcin levels are determined, using standard methodologies asdescribed therein and above. Elevated levels of osteocalcin and alkalinephosphatase are observed in the rats treated with an effective amount ofOP-3. Moreover, histomorphometric analysis on the tibial diasypheal boneshows improved bone mass in OP-3-treated animals as compared withuntreated, ovariectomized rats. In fact, the bone mass of OP-3-animalsis comparable to (e.g., approaches) that of the sham-operated (e.g.,nonovarectomized) rats.

Example 6 Proliferation of Progenitor Cell Populations

Progenitor cells may be stimulated to proliferate in vivo or ex vivo.The cells may be stimulated in vivo by injecting or otherwise providinga sterile preparation containing the morphogenically active fragment ofOP-3 into the individual. For example, the hemopoietic pluripotentialstem cell population of an individual may be stimulated to proliferateby injecting or otherwise providing an appropriate concentration of OP-3to the individual's bone marrow.

Progenitor cells may be stimulated ex vivo by contacting progenitorcells of the population to be enhanced with a morphogenically activefragment of OP-3 under sterile conditions at a concentration and for atime sufficient to stimulate proliferation of the cells. Suitableconcentrations and stimulation times may be determined empirically,essentially following the procedure described in Example 2, above. Amorphogen concentration of between about 0.1-100 ng/ml and a stimulationperiod of from about 10 minutes to about 72 hours, or, more generally,about 24 hours, typically should be sufficient to stimulate a cellpopulation of about 10⁴ to 10⁶ cells. The stimulated cells then areprovided to the individual as, for example, by injecting the cells to anappropriate in vivo locus. Suitable biocompatible progenitor cells maybe obtained by any of the methods known in the art or describedhereinabove.

Example 7 Regeneration of Damaged or Diseased Tissue

OP-3 may be used to repair diseased or damaged mammalian tissue. Thetissue to be repaired preferably is assessed first, and excess necroticor interfering scar tissue removed as needed, e.g., by ablation or bysurgical, chemical, or other methods known in the medical arts.

OP-3 then may be provided directly to the tissue locus as part of asterile, biocompatible composition, either by surgical implantation orinjection. The morphogen also may be provided systemically, as by oralor parenteral administration. Alternatively, a sterile, biocompatiblecomposition containing progenitor cells stimulated by a morphogenicallyactive fragment of OP-3 may be provided to the tissue locus. Theexisting tissue at the locus, whether diseased or damaged, provides theappropriate matrix to allow the proliferation and tissue-specificdifferentiation of progenitor cells. In addition, a damaged or diseasedtissue locus, particularly one that has been further assaulted bysurgical means, provides a morphogenically permissive environment.Systemic provision of OP-3 will be sufficient for certain applications(e.g., in the treatment of osteoporosis and other disorders of the boneremodeling cycle, as an example).

In some circumstances, particularly where tissue damage is extensive,the tissue may not be capable of providing a sufficient matrix for cellinflux and proliferation. In these instances, it may be necessary toprovide OP-3 or progenitor cells stimulated by OP-3 to the tissue locusin association with a suitable, biocompatible, formulated matrix,prepared by any of the means described below. The matrix preferably isin vivo biodegradable. The matrix also may be tissue-specific and/or maycomprise porous particles having dimensions within the range of 70-850μm, most preferably 150-420 μm.

OP-3 also may be used to prevent or substantially inhibitimmune/inflammatory response-mediated tissue damage and scar tissueformation following an injury. OP-3 is provided to a newly injuredtissue locus, to induce tissue morphogenesis at the locus, preventingthe aggregation of migrating fibroblasts into non-differentiatedconnective tissue. OP-3 preferably is provided as a sterilepharmaceutical preparation injected into the tissue locus within fivehours of the injury. Where an immune/inflammatory response isunavoidably or deliberately induced, as part of, for example, a surgicalor other aggressive clinical therapy, OP-3 preferably is providedprophylactically to the patient, prior to, or concomitant with, thetherapy.

Below are several examples, describing protocols for demonstratingOP-3-induced tissue morphogenesis in bone, liver, nerve, dentin,cementum and periodontal tissue.

7.1 OP-3-Induced Bone Morphogenesis

A particularly useful mammalian tissue model system for demonstratingand evaluating the morphogenic activity of a protein is the endochondralbone tissue morphogenesis model known in the art and described, forexample, in U.S. Pat. No. 4,968,590 and incorporated herein byreference. The ability to induce endochondral bone formation includesthe ability to induce the proliferation of progenitor cells intochondroblasts and osteoblasts, the ability to induce cartilage matrixformation, cartilage calcification, and bone remodeling, and the abilityto induce formation of an appropriate vascular supply and hematopoeiticbone marrow differentiation.

The local environment in which the morphogenic material is placed isimportant for tissue morphogenesis. As used herein, "local environment"is understood to include the tissue structural matrix and theenvironment surrounding the tissue. For example, in addition to needingan appropriate anchoring substratum for their proliferation, the cellsstimulated by morphogens need signals to direct the tissue-specificityof their differentiation. These signals vary for the different tissuesand may include cell surface markers. In addition, vascularization ofnew tissue requires a local environment which supports vascularization.

The following sets forth various procedures for evaluating the in vivomorphogenic utility of OP-3 and OP-3-containing compositions. Thecompositions may be injected or surgically implanted in a mammal,following any of a number of procedures well known in the art. Forexample, surgical implant bioassays may be performed essentiallyfollowing the procedure of Sampath et al. (1983) PNAS 80:6591-6595 andU.S. Pat. No. 4,968,590.

Histological sectioning and staining is preferred to determine theextent of morphogenesis in vivo, particularly in tissue repairprocedures. Excised implants are fixed in Bouins Solution, embedded inparaffin, and cut into 6-8 μm sections. Staining with toluidine blue orhemotoxylin/eosin demonstrates clearly the ultimate development of thenew tissue. Twelve day implants are usually sufficient to determinewhether the implants contain newly induced tissue.

Successful implants exhibit a controlled progression through the stagesof induced tissue development allowing one to identify and follow thetissue-specific events that occur. For example, in endochondral boneformation the stages include: (1) leukocytes on day one; (2) mesenchymalcell migration and proliferation on days two and three; (3) chondrocyteappearance on days five and six; (4) cartilage matrix formation on dayseven; (5) cartilage calcification on day eight; (6) vascular invasion,appearance of osteoblasts, and formation of new bone on days nine andten; (7) appearance of osteoclastic cells, and the commencement of boneremodeling and dissolution of the implanted matrix on days twelve toeighteen; and (8) hematopoietic bone marrow differentiation in theresulting ossicles on day twenty-one.

In addition to histological evaluation, biological markers may be usedas markers for tissue morphogenesis. Useful markers includetissue-specific enzymes whose activities may be assayed (e.g.,spectrophotometrically) after homogenization of the implant. Theseassays may be useful for quantitation and for rapidly obtaining anestimate of tissue formation after the implants are removed from theanimal. For example, alkaline phosphatase activity may be used as amarker for osteogenesis.

Incorporation of systemically provided OP-3 may be followed using taggedfragments (e.g., radioactively labelled) and determining theirlocalization in the new tissue, and/or by monitoring their disappearancefrom the circulatory system using a standard labeling protocol andpulse-chase procedure. OP-3 also may be provided with a tissue-specificmolecular tag, whose uptake may be monitored and correlated with theconcentration of OP-3 provided. As an example, ovary removal in femalerats results in reduced bone alkaline phosphatase activity, and rendersthe rats predisposed to osteoporosis (as described in Example 5). If thefemale rats now are provided with OP-3, a reduction in the systemicconcentration of calcium may be seen, which correlates with the presenceof the provided OP-3 and which is anticipated to correspond withincreased alkaline phosphatase activity.

7.2 Morphogen-Induced Liver Regeneration

As another example, a method for inducing morphogenesis of substantiallyinjured liver tissue following a partial hepatectomy utilizing OP-3 ispresented. Variations on this general protocol may be used to testmorphogen activity of OP-3 in other different tissues. The generalmethod involves excising an essentially nonregenerating portion of atissue and providing OP-3, preferably as a soluble pharmaceuticalpreparation to the excised tissue locus, closing the wound, andexamining the site at a future date. Like bone, liver has a potential toregenerate upon injury during post-fetal life.

OP-3, e.g., 1 mg/ml, in a biocompatible solution, for example, (e.g., apurified recombinant mature form of OP-3, is solubilized in 50% ethanol,or compatible solvent, containing 0.1% trifluoroacetic acid, orcompatible acid. Alternatively, the mature protein may be solubilized byassociation with a pro domain. The injectable OP-3 solution is prepared,e.g., by diluting one volume of OP-3 solvent-acid stock solution with 9volumes of 0.2% rat serum albumin in sterile PBS (phosphate-bufferedsaline).

In the experiment, growing rats or aged rats (e.g., Long Evans, CharlesRiver Laboratories, Wilmington) are anesthetized by using ketamine. Twoof the liver lobes (left and right) are cut out (approximately 1/3 ofthe lobe) and the OP-3 is injected locally at multiple sites along thecut ends. The amount of OP-3 injected may be, e.g., 100 μg in 1000 μl ofPBS/RSA (phosphate buffered saline/rat serum albumin) injection buffer.Placebo samples are injection buffer only. In experimental essays, fiverats in each group preferably are used. The wound is closed and the ratsare allowed to eat normal food and drink tap water.

After 12 days, the rats are sacrificed and liver regeneration isobserved visually, to evaluate the effects of the OP-3 on liverregeneration most effectively. The OP-3 fragment-injected group shows,e.g., complete liver tissue regeneration with no sign remaining of anycut in the liver. By contrast, the control group into which only PBS isinjected, shows only minimal regeneration with the incision remaining inthe sample. Previous experiments with other morphogens (e.g., OP-1) showthese morphogens alone induce liver tissue regeneration.

7.3 Morphogen-Induced Dentin, Cementum and Periodontal LigamentRegeneration

As still another example, the ability of OP-3 to induce dentinogenesisalso may be demonstrated. To date, the unpredictable response of dentalpulp tissue to injury is a basic clinical problem in dentistry.Cynomolgus monkeys are chosen as primate models as monkeys are presumedto be more indicative of human dental biology than models based on lowernon-primate mammals.

Using standard dental surgical procedures, small areas (e.g., 2 mm) ofdental pulps are surgically exposed by removing the enamel and dentinimmediately above the pulp (by drilling) of sample teeth, performing apartial amputation of the coronal pulp tissue, inducing hemostasis,application of the pulp treatment, and sealing and filling the cavity bystandard procedures.

Pulp treatments used may include: a morphogenically active fragment ofOP-3 dispersed in a carrier matrix; carrier matrix alone, and notreatment. Twelve teeth per animal (four for each treatment) areprepared, and two animals are used. At four weeks, teeth are extractedand processed histologically for analysis of dentin formation, and/orground to analyze dentin mineralization. The effect of OP-3 onosteodentin reparation may be observed visually by comparing controlsamples treatment (PBS) with OP-3. OP-3 plus a carrier matrix inducesformation of reparative or osteodentin bridges on surgically exposedhealthy dental pulps. By contrast, pulps treated with carrier matrixalone, do not form reparative dentin.

Similarly, implanting demineralized teeth and OP-3 into surgicallyprepared canine tooth sockets stimulates new periodontal tissueformation, including new cementum and periodontal ligament, as well asnew alveolar bone and dentin tissue, as described for OP-1 in U.S. Ser.No. 07/945,285, abandoned, filed on even date herewith, the disclosureof which is incorporated herein by reference. By contrast, untreatedteeth or teeth treated with carrier vehicle alone do not induceperiodontal tissue growth.

7.4 Morphogen-Induced Nerve Tissue Repair

As yet another example, the induction of regenerative effects on centralnervous system (CNS) repair, by a morphogenically active fragment ofOP-3, may be demonstrated using a rat brain stab model. Details of theprotocol are described in copending U.S. Ser. No. 922,813, abandoned.Briefly, male Long Evans rats are anesthetized and the head areaprepared for surgery. The calvariae is exposed using standard surgicalprocedures and a hole drilled toward the center of each lobe using a0.035K wire, just piercing the calvariae. 25 μl solutions containingeither morphogen (e.g., OP-3, 25 μg) or PBS then is provided to each ofthe holes by Hamilton syringe. Solutions are delivered to a depthapproximately 3 mm below the surface, into the underlying cortex, corpuscallosum and hippocampus. The skin then is sutured and the animalallowed to recover.

Three days post surgery, rats are sacrificed by decapitation and theirbrains processed for sectioning. Scar tissue formation is evaluated byimmunofluoresence staining for glial fibrillary acidic protein, a markerprotein for glial scarring, to qualitatively determine the degree ofscar formation. Sections also are probed with OP-3-specific antibody todetermine the presence of the protein. Reduced levels of glialfibrillary acidic protein are observed in the tissue sections of animalstreated with OP-3, evidencing the ability of the morphogen to inhibitglial scar formation, thereby stimulating nerve regeneration.

The ability of OP-3 to stimulate peripheral nervous system axonal growthover extended distances may be demonstrated using the following model.Neurons of the peripheral nervous system can sprout new processes ontheir own following injury, but without guidance these sproutingstypically fail to connect appropriately and die. Where the break isextensive, e.g., greater than 5 or 10 mm, regeneration is poor ornonexistent. Previous experiments with other morphogens, e.g., OP-1,show that morphogens stimulate peripheral nervous system axonal growthover extended distances, allowing repair and regeneration of damagedperipheral neural pathways.

In this example OP-3 stimulation of nerve regeneration is demonstratedusing the rat sciatic nerve model. The rat sciatic nerve can regeneratespontaneously across a 5 mm gap, and occasionally across a 10 mm gap,provided that the severed ends are inserted in a saline-filled nerveguidance channel. In this experiment, nerve regeneration across at leasta 12 mm gap is tested.

Adult female Sprague-Dawley rats (Charles River Laboratories, Inc.)weighing 230-250 g are anesthetized with intraperitoneal injections ofsodium pentobarbital (35 mg/kg body weight). A skin incision is madeparallel and just posterior to the femur. The avascular intermuscularplane between vastus lateralis and hamstring muscles are entered andfollowed to the loose fibroareolar tissue surrounding the sciatic nerve.The loose tissue is divided longitudinally thereby freeing the sciaticnerve over its full extent without devascularizing any portion. Under asurgical microscope the sciatic nerves are transected with microscissorsat mid-thigh and grafted with a OP-3 gel graft that separates the nervestumps by 12 mm. The graft region is encased in a silicone tube 20 mm inlength with a 1.5 mm inner diameter, the interior of which is filledwith the morphogen solution. Specifically, the central 12 mm of the tubeconsists of an OP-3 gel prepared by mixing 1 to 5 μg of substantiallypure recombinantly produced OP-3 protein with approximately 100 μl ofMATRIGEL™ (from Collaborative Research, Inc., Bedford, Mass.), anextracellular matrix extract derived from mouse sarcoma tissue, andcontaining solubilized tissue basement membrane, including laminin, typeIV collagen, heparin sulfate, proteoglycan and entactin, inphosphate-biffered saline. The morphogen-filled tube then is implanteddirectly into the defect site, allowing 4 mm on each end to insert thenerve stumps. Each stump is abutted against the morphogen gel and issecured in the silicone tube by three stitches of commercially availablesurgical 10-0 nylon through the epineurium, the fascicle protectivesheath.

In addition to OP-3 gel grafts, control grafts of empty silicone tubes,silicone tubes filled with gel only and "reverse" autografts, wherein 12mm transected segments of the animal's sciatic nerve are rotated 180°prior to suturing, preferably also are grafted. All experimentspreferably are performed in quadruplicate. All wounds preferably areclosed by wound clips that are removed after 10 days. Rats can begrafted on both legs. At 3 weeks the animals are sacrificed, and thegrafted segments removed and frozen on dry ice immediately. Frozensections then are cut throughout the graft site, and examined for axonalregeneration by immunofluorescent staining using anti-neurofilamentantibodies labeled with flurocein (obtained, for example, from SigmaChemical Co., St. Louis).

Regeneration of the sciatic nerve occurs across the entire 12 mmdistance in all graft sites wherein the gap is filled with the OP-3 gel.By contrast, empty silicone tubes, gel alone and reverse autografts donot show nerve regeneration.

Example 8 Identification of Morphogen-Expressing Tissue

Determining the tissue distribution of morphogens may be used toidentify different morphogens expressed in a given tissue, as well as toidentify new, related morphogens. Tissue distribution also may be usedto identify useful morphogen-producing tissue for use in screening andidentifying candidate morphogen-stimulating agents. The morphogens (ortheir mRNA transcripts) readily are identified in different tissuesusing standard methodologies and minor modifications thereof in tissueswhere expression may be low. For example, protein distribution may bedetermined using standard Western blot analysis or immunofluorescenttechniques, and antibodies specific to the morphogen or morphogens ofinterest. Similarly, the distribution of morphogen transcripts may bedetermined using standard Northern hybridization protocols andtranscript-specific probes.

Any probe capable of hybridizing specifically to a transcript, anddistinguishing the transcript of interest from other, relatedtranscripts may be used. Because the morphogens described herein sharesuch high sequence homology in their active, C-terminal domains, thetissue distribution of a specific morphogen transcript may best bedetermined using a probe specific for the pro region of the immatureprotein and/or the N-terminal region of the mature protein. Anotheruseful sequence is the 3' non-coding region flanking and immediatelyfollowing the stop codon. These portions of the sequence varysubstantially among the morphogens of this invention, and accordingly,are specific for each protein. For example, a particularly usefulOP-3-specific probe sequence is one derived from a portion of the 3'untranslated sequence, e.g., nucleotides 1310-1674 of Seq. ID No. 1,which shares little or no homology with other morphogen sequences,including OP-2. The chosen fragment then is labelled using standardmeans well known and described in the art.

Using these morphogen-specific probes, which may be syntheticallyengineered or obtained from cloned sequences, morphogen transcripts canbe identified in mammalian tissue, using standard methodologies wellknown to those having ordinary skill in the art. A detailed descriptionof a suitable hybridization protocol is described in Ozkaynak, et al.,(1991) Biochem. Biophys. Res. Commn. 179:116-123, and Ozkaynak, etal.(1992) (JBC, in press), the disclosure of which are incorporatedherein by reference. Briefly, total RNA is prepared from various tissues(e.g., murine embryo and developing and adult liver, kidney, testis,heart, brain, thymus, stomach) by a standard methodology such as by themethod of Chomczyaski et al. ((1987) Anal. Biochem 162:156-159) anddescribed below. Poly (A)+RNA is prepared by using oligo (dT)-cellulosechromatography (e.g., Type 7, from Pharmacia LKB Biotechnology, Inc.).Poly (A)+RNA (generally 15 μg) from each tissue is fractionated on a 1%agarose/formaldehyde gel and transferred onto a Nytran membrane(Schleicher & Schuell). Following the transfer, the membrane is baked at80° C. and the RNA is cross-linked under UV light (generally 30 secondsat 1 mW/cm²). Prior to hybridization, the appropriate probe is denaturedby heating. The hybridization is carried out in a lucite cylinderrotating in a roller bottle apparatus at approximately 1 rev/min forapproximately 15 hours at 37° C. using a hybridization mix of 40%formamide, 55× Denhardts, 5× SSPE, and 0.1% SDS. Followinghybridization, the non-specific counts are washed off the filters in0.1× SSPE, 0.1% SDS at 50° C.

An OP-3-specific 0.5 kb probe was made from a StuI-BglII fragment ofOP-3 cDNA. The fragment contains the 3' untranslated sequence fromnucleotides 1310-1674, plus an additional 140 bases. The fragment waslabelled using standard techniques and the hybridization performed asdescribed. To date, OP-3, like OP-2, appears to be expressed primarilyin early embryonic tissue. Specifically, Northern blots of murineembryos show abundant OP-3 expression in 8-day embryos, demonstrated bya strong band at 2.9 kb and a weaker band at 2.3 kb.

Example 9 Screening Assay for Candidate Compounds which Alter EndogenousMorphogen Levels

Candidate compound(s) which may be administered to affect the level ofendogenous OP-3 morphogen may be found using the following screeningassay, in which the level of OP-3 production by a cell type whichproduces measurable levels of the morphogen is determined with andwithout incubating the cell in culture with the compound, in order toassess the effects of the compound on the cell. This can be accomplishedby detection of the morphogen either at the protein or RNA level. Adetailed description also may be found in U.S. Ser. No. 752,861,abandoned, and U.S. Ser. No. 938,021, abandoned, incorporatedhereinabove by reference.

9.1 Growth of Cells in Culture

Cell cultures of kidney, adrenals, urinary bladder, brain, or otherorgans, may be prepared as described widely in the literature. Forexample, kidneys may be explanted from neonatal or new born or young oradult rodents (mouse or rat) and used in organ culture as whole orsliced (1-4 mm) tissues. Primary tissue cultures and established celllines, also derived from kidney, adrenals, urinary, bladder, brain,mammary, or other tissues may be established in multiwell plates (6 wellor 24 well) according to conventional cell culture techniques, and arecultured in the absence or presence of serum for a period of time (1-7days). Cells may be cultured, for example, in Dulbecco's Modified Eaglemedium (Gibco, Long Island, N.Y.) containing serum (e.g., fetal calfserum at 1%-10%, Gibco) or in serum-deprived medium, as desired, or indefined medium (e.g., containing insulin, transferrin, glucose, albumin,or other growth factors).

Samples for testing the level of morphogen production includes culturesupernatants or cell lysates, collected periodically and evaluated formorphogen production by immunoblot analysis (Sambrook et al., eds.,1989, Molecular Cloning, Cold Spring Harbor Press, Cold Spring Harbor,N.Y.), or a portion of the cell culture itself, collected periodicallyand used to prepare polyA+ RNA for RNA analysis. To monitor de novomorphogen synthesis, some cultures are labeled according to conventionalprocedures with an ³⁵ S-methionine/ ³⁵ S-cysteine mixture for 6-24 hoursand then evaluated for morphogenic protein synthesis by conventionalimmunoprecipitation methods.

9.2 Determination of Level of Morphogenic Protein

In order to quantitate the production of a morphogenic protein, e.g.,OP-3, by a cell type, an immunoassay may be performed to detect themorphogen using a polyclonal or monoclonal antibody specific for thatprotein. For example, OP-3 may be detected using a polyclonal antibodyspecific for OP-3 in an ELISA, as follows.

1 μg/100 μl of affinity-purified polyclonal rabbit IgG specific for OP-3is added to each well of a 96-well plate and incubated at 37° C. for anhour. The wells are washed four times with 0.167M sodium borate bufferwith 0.15M NaCl (BSB), pH 8.2, containing 0.1% Tween 20. To minimizenon-specific binding, the wells are blocked by filling completely with1% bovine serum albumin (BSA) in BSB and incubating for 1 hour at 37° C.The wells are then washed four times with BSB containing 0.1% Tween 20.A 100 μl aliquot of an appropriate dilution of each of the test samplesof cell culture supernatant is added to each well in triplicate andincubated at 37° C. for 30 min. After incubation, 100 μl biotinylatedrabbit anti-OP-3 serum (stock solution is about 1 mg/ml and diluted1:400 in BSB containing 1% BSA before use) is added to each well andincubated at 37° C. for 30 min. The wells are then washed four timeswith BSB containing 0.1% Tween 20. 100 μl strepavidin-alkaline (SouthernBiotechnology Associates, Inc. Birmingham, Ala., diluted 1:2000 in BSBcontaining 0.1% Tween 20 before use) is added to each well and incubatedat 37° C. for 30 min. The plates are washed four times with 0.5M Trisbuffered Saline (TBS), pH 7.2. 50 μl substrate (ELISA AmplificationSystem Kit, Life Technologies, Inc., Bethesda, Md.) is added to eachwell incubated at room temperature for 15 min. Then, 50 μl amplifier(from the same amplification system kit) is added and incubated foranother 15 min at room temperature. The reaction is stopped by theaddition of 50 μl 0.3M sulphuric acid. The OD at 490 nm of the solutionin each well is recorded. To quantitate OP-3 in culture media, an OP-3standard curve is performed in parallel with the test samples.

Polyclonal antibody may be prepared as follows. Each rabbit is given aprimary immunization of 100 ug/500 μl recombinantly-produced OP-3protein or protein fragment in 0.1% SDS mixed with 500 μl CompleteFreund's Adjuvant. The antigen is injected subcutaneously at multiplesites on the back and flanks of the animal. The rabbit is boosted aftera month in the same manner using incomplete Freund's Adjuvant. Testbleeds are taken from the ear vein seven days later. Two additionalboosts and test bleeds are performed at monthly intervals until antibodyagainst OP-3 is detected in the serum using an ELISA assay. Then, therabbit is boosted monthly with 100 μg of antigen and bled (15 ml perbleed) at days seven and ten after boosting.

Monoclonal antibody specific for a given morphogen may be prepared asfollows. A mouse is given two injections of OP-3 protein or a proteinfragment specific for OP-3. The protein preferably is recombinantlyproduced. The first injection contains 100 μg of OP-3 in completeFreund's adjuvant and is given subcutaneously. The second injectioncontains 50 μg of OP-3 in incomplete adjuvant and is givenintraperitoneally. The mouse then receives a total of 230 μg of OP-3 infour intraperitoneal injections at various times over an eight monthperiod. One week prior to fusion, the mouse is boosted intraperitoneallywith OP-3 (e.g., 100 μg) and may be additionally boosted with anOP-3-specific peptide (e.g., corresponding to the N-terminus of themature protein) conjugated to bovine serum albumin with a suitablecross-linking agent. This boost can be repeated five days (IP), fourdays (IP), three days (IP) and one day (IV) prior to fusion. The mousespleen cells then are fused to commercially available myeloma cells at aratio of 1:1 using PEG 1500 (Boeringer Mannheim, Germany), and the fusedcells plated and screened for OP-3-specific antibodies using OP-3 asantigen. The cell fusion and monoclonal screening steps readily areperformed according to standard procedures well described in standardtexts widely available in the art.

Other Embodiments

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The presentembodiments are therefore to be considered in all respects asillustrative and not restrictive, the scope of the invention beingindicated by the appended claims rather than by the foregoingdescription, and all changes which come within the meaning and range ofequivalency of the claims are therefore intended to be embraced therein.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 13    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1674 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 69..1265    (D) OTHER INFORMATION: /note= "mOP3-PP"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    GGATCCGCGGCGCTGTCCCATCCTTGTCGTCGAGGCGTCGCTGGATGCGAGTCCGCTAAA60    CGTCCGAGATGGCTGCGCGTCCGGGACTCCTATGGCTACTGGGCCTGGCT110    MetAlaAlaArgProGlyLeuLeuTrpLeuLeuGlyLeuAla    1510    CTGTGCGTGTTGGGCGGCGGTCACCTCTCGCATCCCCCGCACGTCTTT158    LeuCysValLeuGlyGlyGlyHisLeuSerHisProProHisValPhe    15202530    CCCCAGCGTCGACTAGGAGTACGCGAGCCCCGCGACATGCAGCGCGAG206    ProGlnArgArgLeuGlyValArgGluProArgAspMetGlnArgGlu    354045    ATTCGGGAGGTGCTGGGGCTAGCCGGGCGGCCCCGATCCCGAGCACCG254    IleArgGluValLeuGlyLeuAlaGlyArgProArgSerArgAlaPro    505560    GTCGGGGCTGCCCAGCAGCCAGCGTCTGCGCCCCTCTTTATGTTGGAC302    ValGlyAlaAlaGlnGlnProAlaSerAlaProLeuPheMetLeuAsp    657075    CTGTACCGTGCCATGACGGATGACAGTGGCGGTGGGACCCCGCAGCCT350    LeuTyrArgAlaMetThrAspAspSerGlyGlyGlyThrProGlnPro    808590    CACTTGGACCGTGCTGACCTGATTATGAGCTTTGTCAACATAGTGGAA398    HisLeuAspArgAlaAspLeuIleMetSerPheValAsnIleValGlu    95100105110    CGCGACCGTACCCTGGGCTACCAGGAGCCACACTGGAAGGAATTCCAC446    ArgAspArgThrLeuGlyTyrGlnGluProHisTrpLysGluPheHis    115120125    TTTGACCTAACCCAGATCCCTGCTGGGGAGGCTGTCACAGCTGCTGAG494    PheAspLeuThrGlnIleProAlaGlyGluAlaValThrAlaAlaGlu    130135140    TTCCGGATCTACAAAGAACCCAGTACCCACCCGCTCAACACAACCCTC542    PheArgIleTyrLysGluProSerThrHisProLeuAsnThrThrLeu    145150155    CACATCAGCATGTTCGAAGTGGTCCAAGAGCACTCCAACAGGGAGTCT590    HisIleSerMetPheGluValValGlnGluHisSerAsnArgGluSer    160165170    GACTTGTTCTTTTTGGATCTTCAGACGCTCCGATCTGGGGACGAGGGC638    AspLeuPhePheLeuAspLeuGlnThrLeuArgSerGlyAspGluGly    175180185190    TGGCTGGTGCTGGACATCACAGCAGCCAGTGACCGATGGCTGCTGAAC686    TrpLeuValLeuAspIleThrAlaAlaSerAspArgTrpLeuLeuAsn    195200205    CATCACAAGGACCTAGGACTCCGCCTCTATGTGGAAACCGAGGATGGG734    HisHisLysAspLeuGlyLeuArgLeuTyrValGluThrGluAspGly    210215220    CACAGCATAGATCCTGGCCTAGCTGGTCTGCTTGGACGACAAGCACCA782    HisSerIleAspProGlyLeuAlaGlyLeuLeuGlyArgGlnAlaPro    225230235    CGCTCCAGACAGCCTTTCATGGTTGGTTTCTTCAGGGCCAACCAGAGT830    ArgSerArgGlnProPheMetValGlyPhePheArgAlaAsnGlnSer    240245250    CCTGTGCGGGCCCCTCGAACAGCAAGACCACTGAAGAAGAAGCAGCTA878    ProValArgAlaProArgThrAlaArgProLeuLysLysLysGlnLeu    255260265270    AATCAAATCAACCAGCTGCCGCACTCCAACAAACACCTAGGAATCCTT926    AsnGlnIleAsnGlnLeuProHisSerAsnLysHisLeuGlyIleLeu    275280285    GATGATGGCCACGGTTCTCACGGCAGAGAAGTTTGCCGCAGGCATGAG974    AspAspGlyHisGlySerHisGlyArgGluValCysArgArgHisGlu    290295300    CTCTATGTCAGCTTCCGTGACCTTGGCTGGCTGGACTCTGTCATTGCC1022    LeuTyrValSerPheArgAspLeuGlyTrpLeuAspSerValIleAla    305310315    CCCCAGGGCTACTCCGCCTATTACTGTGCTGGGGAGTGCATCTACCCA1070    ProGlnGlyTyrSerAlaTyrTyrCysAlaGlyGluCysIleTyrPro    320325330    CTGAACTCCTGTATGAACTCCACCAACCACGCCACTATGCAGGCCCTG1118    LeuAsnSerCysMetAsnSerThrAsnHisAlaThrMetGlnAlaLeu    335340345350    GTACATCTGATGAAGCCAGATATCATCCCCAAGGTGTGCTGTGTGCCT1166    ValHisLeuMetLysProAspIleIleProLysValCysCysValPro    355360365    ACTGAGCTGAGTGCCATTTCTCTGCTCTACTATGATAGAAACAATAAT1214    ThrGluLeuSerAlaIleSerLeuLeuTyrTyrAspArgAsnAsnAsn    370375380    GTCATCCTGCGCAGGGAGCGCAACATGGTAGTCCAGGCCTGTGGCTGC1262    ValIleLeuArgArgGluArgAsnMetValValGlnAlaCysGlyCys    385390395    CACTGAGTCCCTGCCCAACAGCCTGCTGCCATCCCATCTATCTAGTCAGGCCT1315    His    CTCTTCCAAGGCAGGAAACCAACAAAGAGGGAAGGCAGTGCTTTCAACTCCATGTCCACA1375    TTCACAGTCTTGGCCCTCTCTGTTCTTTTTGCCAAGGCTGAGAAGATGGTCCTAGTTATA1435    ACCCTGGTGACCTCAGTAGCCCGATCTCTCATCTCCCCAAACTCCCCAATGCAGCCAGGG1495    GCATCTATGTCCTTTGGGATTGGGCACAGAAGTCCAATTTACCAACTTATTCATGAGTCA1555    CTACTGGCCCAGCCTGGACTTGAACCTGGAACACAGGGTAGAGCTCAGGCTCTTCAGTAT1615    CCATCAGAAGATTTAGGTGTGTGCAGACATGACCACACTCCCCCTAGCACTCCATAGCC1674    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 399 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    MetAlaAlaArgProGlyLeuLeuTrpLeuLeuGlyLeuAlaLeuCys    151015    ValLeuGlyGlyGlyHisLeuSerHisProProHisValPheProGln    202530    ArgArgLeuGlyValArgGluProArgAspMetGlnArgGluIleArg    354045    GluValLeuGlyLeuAlaGlyArgProArgSerArgAlaProValGly    505560    AlaAlaGlnGlnProAlaSerAlaProLeuPheMetLeuAspLeuTyr    65707580    ArgAlaMetThrAspAspSerGlyGlyGlyThrProGlnProHisLeu    859095    AspArgAlaAspLeuIleMetSerPheValAsnIleValGluArgAsp    100105110    ArgThrLeuGlyTyrGlnGluProHisTrpLysGluPheHisPheAsp    115120125    LeuThrGlnIleProAlaGlyGluAlaValThrAlaAlaGluPheArg    130135140    IleTyrLysGluProSerThrHisProLeuAsnThrThrLeuHisIle    145150155160    SerMetPheGluValValGlnGluHisSerAsnArgGluSerAspLeu    165170175    PhePheLeuAspLeuGlnThrLeuArgSerGlyAspGluGlyTrpLeu    180185190    ValLeuAspIleThrAlaAlaSerAspArgTrpLeuLeuAsnHisHis    195200205    LysAspLeuGlyLeuArgLeuTyrValGluThrGluAspGlyHisSer    210215220    IleAspProGlyLeuAlaGlyLeuLeuGlyArgGlnAlaProArgSer    225230235240    ArgGlnProPheMetValGlyPhePheArgAlaAsnGlnSerProVal    245250255    ArgAlaProArgThrAlaArgProLeuLysLysLysGlnLeuAsnGln    260265270    IleAsnGlnLeuProHisSerAsnLysHisLeuGlyIleLeuAspAsp    275280285    GlyHisGlySerHisGlyArgGluValCysArgArgHisGluLeuTyr    290295300    ValSerPheArgAspLeuGlyTrpLeuAspSerValIleAlaProGln    305310315320    GlyTyrSerAlaTyrTyrCysAlaGlyGluCysIleTyrProLeuAsn    325330335    SerCysMetAsnSerThrAsnHisAlaThrMetGlnAlaLeuValHis    340345350    LeuMetLysProAspIleIleProLysValCysCysValProThrGlu    355360365    LeuSerAlaIleSerLeuLeuTyrTyrAspArgAsnAsnAsnValIle    370375380    LeuArgArgGluArgAsnMetValValGlnAlaCysGlyCysHis    385390395    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1822 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 49..1341    (D) OTHER INFORMATION: /function="OSTEOGENIC PROTEIN"    /product= "hOP1-PP"    /note= "hOP1 cDNA"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    GGTGCGGGCCCGGAGCCCGGAGCCCGGGTAGCGCGTAGAGCCGGCGCGATGCACGTG57    MetHisVal    CGCTCACTGCGAGCTGCGGCGCCGCACAGCTTCGTGGCGCTCTGGGCA105    ArgSerLeuArgAlaAlaAlaProHisSerPheValAlaLeuTrpAla    51015    CCCCTGTTCCTGCTGCGCTCCGCCCTGGCCGACTTCAGCCTGGACAAC153    ProLeuPheLeuLeuArgSerAlaLeuAlaAspPheSerLeuAspAsn    20253035    GAGGTGCACTCGAGCTTCATCCACCGGCGCCTCCGCAGCCAGGAGCGG201    GluValHisSerSerPheIleHisArgArgLeuArgSerGlnGluArg    404550    CGGGAGATGCAGCGCGAGATCCTCTCCATTTTGGGCTTGCCCCACCGC249    ArgGluMetGlnArgGluIleLeuSerIleLeuGlyLeuProHisArg    556065    CCGCGCCCGCACCTCCAGGGCAAGCACAACTCGGCACCCATGTTCATG297    ProArgProHisLeuGlnGlyLysHisAsnSerAlaProMetPheMet    707580    CTGGACCTGTACAACGCCATGGCGGTGGAGGAGGGCGGCGGGCCCGGC345    LeuAspLeuTyrAsnAlaMetAlaValGluGluGlyGlyGlyProGly    859095    GGCCAGGGCTTCTCCTACCCCTACAAGGCCGTCTTCAGTACCCAGGGC393    GlyGlnGlyPheSerTyrProTyrLysAlaValPheSerThrGlnGly    100105110115    CCCCCTCTGGCCAGCCTGCAAGATAGCCATTTCCTCACCGACGCCGAC441    ProProLeuAlaSerLeuGlnAspSerHisPheLeuThrAspAlaAsp    120125130    ATGGTCATGAGCTTCGTCAACCTCGTGGAACATGACAAGGAATTCTTC489    MetValMetSerPheValAsnLeuValGluHisAspLysGluPhePhe    135140145    CACCCACGCTACCACCATCGAGAGTTCCGGTTTGATCTTTCCAAGATC537    HisProArgTyrHisHisArgGluPheArgPheAspLeuSerLysIle    150155160    CCAGAAGGGGAAGCTGTCACGGCAGCCGAATTCCGGATCTACAAGGAC585    ProGluGlyGluAlaValThrAlaAlaGluPheArgIleTyrLysAsp    165170175    TACATCCGGGAACGCTTCGACAATGAGACGTTCCGGATCAGCGTTTAT633    TyrIleArgGluArgPheAspAsnGluThrPheArgIleSerValTyr    180185190195    CAGGTGCTCCAGGAGCACTTGGGCAGGGAATCGGATCTCTTCCTGCTC681    GlnValLeuGlnGluHisLeuGlyArgGluSerAspLeuPheLeuLeu    200205210    GACAGCCGTACCCTCTGGGCCTCGGAGGAGGGCTGGCTGGTGTTTGAC729    AspSerArgThrLeuTrpAlaSerGluGluGlyTrpLeuValPheAsp    215220225    ATCACAGCCACCAGCAACCACTGGGTGGTCAATCCGCGGCACAACCTG777    IleThrAlaThrSerAsnHisTrpValValAsnProArgHisAsnLeu    230235240    GGCCTGCAGCTCTCGGTGGAGACGCTGGATGGGCAGAGCATCAACCCC825    GlyLeuGlnLeuSerValGluThrLeuAspGlyGlnSerIleAsnPro    245250255    AAGTTGGCGGGCCTGATTGGGCGGCACGGGCCCCAGAACAAGCAGCCC873    LysLeuAlaGlyLeuIleGlyArgHisGlyProGlnAsnLysGlnPro    260265270275    TTCATGGTGGCTTTCTTCAAGGCCACGGAGGTCCACTTCCGCAGCATC921    PheMetValAlaPhePheLysAlaThrGluValHisPheArgSerIle    280285290    CGGTCCACGGGGAGCAAACAGCGCAGCCAGAACCGCTCCAAGACGCCC969    ArgSerThrGlySerLysGlnArgSerGlnAsnArgSerLysThrPro    295300305    AAGAACCAGGAAGCCCTGCGGATGGCCAACGTGGCAGAGAACAGCAGC1017    LysAsnGlnGluAlaLeuArgMetAlaAsnValAlaGluAsnSerSer    310315320    AGCGACCAGAGGCAGGCCTGTAAGAAGCACGAGCTGTATGTCAGCTTC1065    SerAspGlnArgGlnAlaCysLysLysHisGluLeuTyrValSerPhe    325330335    CGAGACCTGGGCTGGCAGGACTGGATCATCGCGCCTGAAGGCTACGCC1113    ArgAspLeuGlyTrpGlnAspTrpIleIleAlaProGluGlyTyrAla    340345350355    GCCTACTACTGTGAGGGGGAGTGTGCCTTCCCTCTGAACTCCTACATG1161    AlaTyrTyrCysGluGlyGluCysAlaPheProLeuAsnSerTyrMet    360365370    AACGCCACCAACCACGCCATCGTGCAGACGCTGGTCCACTTCATCAAC1209    AsnAlaThrAsnHisAlaIleValGlnThrLeuValHisPheIleAsn    375380385    CCGGAAACGGTGCCCAAGCCCTGCTGTGCGCCCACGCAGCTCAATGCC1257    ProGluThrValProLysProCysCysAlaProThrGlnLeuAsnAla    390395400    ATCTCCGTCCTCTACTTCGATGACAGCTCCAACGTCATCCTGAAGAAA1305    IleSerValLeuTyrPheAspAspSerSerAsnValIleLeuLysLys    405410415    TACAGAAACATGGTGGTCCGGGCCTGTGGCTGCCACTAGCTCCTCC1351    TyrArgAsnMetValValArgAlaCysGlyCysHis    420425430    GAGAATTCAGACCCTTTGGGGCCAAGTTTTTCTGGATCCTCCATTGCTCGCCTTGGCCAG1411    GAACCAGCAGACCAACTGCCTTTTGTGAGACCTTCCCCTCCCTATCCCCAACTTTAAAGG1471    TGTGAGAGTATTAGGAAACATGAGCAGCATATGGCTTTTGATCAGTTTTTCAGTGGCAGC1531    ATCCAATGAACAAGATCCTACAAGCTGTGCAGGCAAAACCTAGCAGGAAAAAAAAACAAC1591    GCATAAAGAAAAATGGCCGGGCCAGGTCATTGGCTGGGAAGTCTCAGCCATGCACGGACT1651    CGTTTCCAGAGGTAATTATGAGCGCCTACCAGCCAGGCCACCCAGCCGTGGGAGGAAGGG1711    GGCGTGGCAAGGGGTGGGCACATTGGTGTCTGTGCGAAAGGAAAATTGACCCGGAAGTTC1771    CTGTAATAAATGTCACAATAAAACGAATGAATGAAAAAAAAAAAAAAAAAA1822    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 431 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    MetHisValArgSerLeuArgAlaAlaAlaProHisSerPheValAla    151015    LeuTrpAlaProLeuPheLeuLeuArgSerAlaLeuAlaAspPheSer    202530    LeuAspAsnGluValHisSerSerPheIleHisArgArgLeuArgSer    354045    GlnGluArgArgGluMetGlnArgGluIleLeuSerIleLeuGlyLeu    505560    ProHisArgProArgProHisLeuGlnGlyLysHisAsnSerAlaPro    65707580    MetPheMetLeuAspLeuTyrAsnAlaMetAlaValGluGluGlyGly    859095    GlyProGlyGlyGlnGlyPheSerTyrProTyrLysAlaValPheSer    100105110    ThrGlnGlyProProLeuAlaSerLeuGlnAspSerHisPheLeuThr    115120125    AspAlaAspMetValMetSerPheValAsnLeuValGluHisAspLys    130135140    GluPhePheHisProArgTyrHisHisArgGluPheArgPheAspLeu    145150155160    SerLysIleProGluGlyGluAlaValThrAlaAlaGluPheArgIle    165170175    TyrLysAspTyrIleArgGluArgPheAspAsnGluThrPheArgIle    180185190    SerValTyrGlnValLeuGlnGluHisLeuGlyArgGluSerAspLeu    195200205    PheLeuLeuAspSerArgThrLeuTrpAlaSerGluGluGlyTrpLeu    210215220    ValPheAspIleThrAlaThrSerAsnHisTrpValValAsnProArg    225230235240    HisAsnLeuGlyLeuGlnLeuSerValGluThrLeuAspGlyGlnSer    245250255    IleAsnProLysLeuAlaGlyLeuIleGlyArgHisGlyProGlnAsn    260265270    LysGlnProPheMetValAlaPhePheLysAlaThrGluValHisPhe    275280285    ArgSerIleArgSerThrGlySerLysGlnArgSerGlnAsnArgSer    290295300    LysThrProLysAsnGlnGluAlaLeuArgMetAlaAsnValAlaGlu    305310315320    AsnSerSerSerAspGlnArgGlnAlaCysLysLysHisGluLeuTyr    325330335    ValSerPheArgAspLeuGlyTrpGlnAspTrpIleIleAlaProGlu    340345350    GlyTyrAlaAlaTyrTyrCysGluGlyGluCysAlaPheProLeuAsn    355360365    SerTyrMetAsnAlaThrAsnHisAlaIleValGlnThrLeuValHis    370375380    PheIleAsnProGluThrValProLysProCysCysAlaProThrGln    385390395400    LeuAsnAlaIleSerValLeuTyrPheAspAspSerSerAsnValIle    405410415    LeuLysLysTyrArgAsnMetValValArgAlaCysGlyCysHis    420425430    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1873 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 104..1393    (D) OTHER INFORMATION: /function="OSTEOGENIC PROTEIN"    /product= "mOP1-PP"    /note= "mOP1 cDNA"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    CTGCAGCAAGTGACCTCGGGTCGTGGACCGCTGCCCTGCCCCCTCCGCTGCCACCTGGGG60    CGGCGCGGGCCCGGTGCCCCGGATCGCGCGTAGAGCCGGCGCGATGCACGTGCGC115    MetHisValArg    1    TCGCTGCGCGCTGCGGCGCCACACAGCTTCGTGGCGCTCTGGGCGCCT163    SerLeuArgAlaAlaAlaProHisSerPheValAlaLeuTrpAlaPro    5101520    CTGTTCTTGCTGCGCTCCGCCCTGGCCGATTTCAGCCTGGACAACGAG211    LeuPheLeuLeuArgSerAlaLeuAlaAspPheSerLeuAspAsnGlu    253035    GTGCACTCCAGCTTCATCCACCGGCGCCTCCGCAGCCAGGAGCGGCGG259    ValHisSerSerPheIleHisArgArgLeuArgSerGlnGluArgArg    404550    GAGATGCAGCGGGAGATCCTGTCCATCTTAGGGTTGCCCCATCGCCCG307    GluMetGlnArgGluIleLeuSerIleLeuGlyLeuProHisArgPro    556065    CGCCCGCACCTCCAGGGAAAGCATAATTCGGCGCCCATGTTCATGTTG355    ArgProHisLeuGlnGlyLysHisAsnSerAlaProMetPheMetLeu    707580    GACCTGTACAACGCCATGGCGGTGGAGGAGAGCGGGCCGGACGGACAG403    AspLeuTyrAsnAlaMetAlaValGluGluSerGlyProAspGlyGln    859095100    GGCTTCTCCTACCCCTACAAGGCCGTCTTCAGTACCCAGGGCCCCCCT451    GlyPheSerTyrProTyrLysAlaValPheSerThrGlnGlyProPro    105110115    TTAGCCAGCCTGCAGGACAGCCATTTCCTCACTGACGCCGACATGGTC499    LeuAlaSerLeuGlnAspSerHisPheLeuThrAspAlaAspMetVal    120125130    ATGAGCTTCGTCAACCTAGTGGAACATGACAAAGAATTCTTCCACCCT547    MetSerPheValAsnLeuValGluHisAspLysGluPhePheHisPro    135140145    CGATACCACCATCGGGAGTTCCGGTTTGATCTTTCCAAGATCCCCGAG595    ArgTyrHisHisArgGluPheArgPheAspLeuSerLysIleProGlu    150155160    GGCGAACGGGTGACCGCAGCCGAATTCAGGATCTATAAGGACTACATC643    GlyGluArgValThrAlaAlaGluPheArgIleTyrLysAspTyrIle    165170175180    CGGGAGCGATTTGACAACGAGACCTTCCAGATCACAGTCTATCAGGTG691    ArgGluArgPheAspAsnGluThrPheGlnIleThrValTyrGlnVal    185190195    CTCCAGGAGCACTCAGGCAGGGAGTCGGACCTCTTCTTGCTGGACAGC739    LeuGlnGluHisSerGlyArgGluSerAspLeuPheLeuLeuAspSer    200205210    CGCACCATCTGGGCTTCTGAGGAGGGCTGGTTGGTGTTTGATATCACA787    ArgThrIleTrpAlaSerGluGluGlyTrpLeuValPheAspIleThr    215220225    GCCACCAGCAACCACTGGGTGGTCAACCCTCGGCACAACCTGGGCTTA835    AlaThrSerAsnHisTrpValValAsnProArgHisAsnLeuGlyLeu    230235240    CAGCTCTCTGTGGAGACCCTGGATGGGCAGAGCATCAACCCCAAGTTG883    GlnLeuSerValGluThrLeuAspGlyGlnSerIleAsnProLysLeu    245250255260    GCAGGCCTGATTGGACGGCATGGACCCCAGAACAAGCAACCCTTCATG931    AlaGlyLeuIleGlyArgHisGlyProGlnAsnLysGlnProPheMet    265270275    GTGGCCTTCTTCAAGGCCACGGAAGTCCATCTCCGTAGTATCCGGTCC979    ValAlaPhePheLysAlaThrGluValHisLeuArgSerIleArgSer    280285290    ACGGGGGGCAAGCAGCGCAGCCAGAATCGCTCCAAGACGCCAAAGAAC1027    ThrGlyGlyLysGlnArgSerGlnAsnArgSerLysThrProLysAsn    295300305    CAAGAGGCCCTGAGGATGGCCAGTGTGGCAGAAAACAGCAGCAGTGAC1075    GlnGluAlaLeuArgMetAlaSerValAlaGluAsnSerSerSerAsp    310315320    CAGAGGCAGGCCTGCAAGAAACATGAGCTGTACGTCAGCTTCCGAGAC1123    GlnArgGlnAlaCysLysLysHisGluLeuTyrValSerPheArgAsp    325330335340    CTTGGCTGGCAGGACTGGATCATTGCACCTGAAGGCTATGCTGCCTAC1171    LeuGlyTrpGlnAspTrpIleIleAlaProGluGlyTyrAlaAlaTyr    345350355    TACTGTGAGGGAGAGTGCGCCTTCCCTCTGAACTCCTACATGAACGCC1219    TyrCysGluGlyGluCysAlaPheProLeuAsnSerTyrMetAsnAla    360365370    ACCAACCACGCCATCGTCCAGACACTGGTTCACTTCATCAACCCAGAC1267    ThrAsnHisAlaIleValGlnThrLeuValHisPheIleAsnProAsp    375380385    ACAGTACCCAAGCCCTGCTGTGCGCCCACCCAGCTCAACGCCATCTCT1315    ThrValProLysProCysCysAlaProThrGlnLeuAsnAlaIleSer    390395400    GTCCTCTACTTCGACGACAGCTCTAATGTCATCCTGAAGAAGTACAGA1363    ValLeuTyrPheAspAspSerSerAsnValIleLeuLysLysTyrArg    405410415420    AACATGGTGGTCCGGGCCTGTGGCTGCCACTAGCTCTTCCTGAGACCCTG1413    AsnMetValValArgAlaCysGlyCysHis    425430    ACCTTTGCGGGGCCACACCTTTCCAAATCTTCGATGTCTCACCATCTAAGTCTCTCACTG1473    CCCACCTTGGCGAGGAGAACAGACCAACCTCTCCTGAGCCTTCCCTCACCTCCCAACCGG1533    AAGCATGTAAGGGTTCCAGAAACCTGAGCGTGCAGCAGCTGATGAGCGCCCTTTCCTTCT1593    GGCACGTGACGGACAAGATCCTACCAGCTACCACAGCAAACGCCTAAGAGCAGGAAAAAT1653    GTCTGCCAGGAAAGTGTCCAGTGTCCACATGGCCCCTGGCGCTCTGAGTCTTTGAGGAGT1713    AATCGCAAGCCTCGTTCAGCTGCAGCAGAAGGAAGGGCTTAGCCAGGGTGGGCGCTGGCG1773    TCTGTGTTGAAGGGAAACCAAGCAGAAGCCACTGTAATGATATGTCACAATAAAACCCAT1833    GAATGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAATTC1873    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 430 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    MetHisValArgSerLeuArgAlaAlaAlaProHisSerPheValAla    151015    LeuTrpAlaProLeuPheLeuLeuArgSerAlaLeuAlaAspPheSer    202530    LeuAspAsnGluValHisSerSerPheIleHisArgArgLeuArgSer    354045    GlnGluArgArgGluMetGlnArgGluIleLeuSerIleLeuGlyLeu    505560    ProHisArgProArgProHisLeuGlnGlyLysHisAsnSerAlaPro    65707580    MetPheMetLeuAspLeuTyrAsnAlaMetAlaValGluGluSerGly    859095    ProAspGlyGlnGlyPheSerTyrProTyrLysAlaValPheSerThr    100105110    GlnGlyProProLeuAlaSerLeuGlnAspSerHisPheLeuThrAsp    115120125    AlaAspMetValMetSerPheValAsnLeuValGluHisAspLysGlu    130135140    PhePheHisProArgTyrHisHisArgGluPheArgPheAspLeuSer    145150155160    LysIleProGluGlyGluArgValThrAlaAlaGluPheArgIleTyr    165170175    LysAspTyrIleArgGluArgPheAspAsnGluThrPheGlnIleThr    180185190    ValTyrGlnValLeuGlnGluHisSerGlyArgGluSerAspLeuPhe    195200205    LeuLeuAspSerArgThrIleTrpAlaSerGluGluGlyTrpLeuVal    210215220    PheAspIleThrAlaThrSerAsnHisTrpValValAsnProArgHis    225230235240    AsnLeuGlyLeuGlnLeuSerValGluThrLeuAspGlyGlnSerIle    245250255    AsnProLysLeuAlaGlyLeuIleGlyArgHisGlyProGlnAsnLys    260265270    GlnProPheMetValAlaPhePheLysAlaThrGluValHisLeuArg    275280285    SerIleArgSerThrGlyGlyLysGlnArgSerGlnAsnArgSerLys    290295300    ThrProLysAsnGlnGluAlaLeuArgMetAlaSerValAlaGluAsn    305310315320    SerSerSerAspGlnArgGlnAlaCysLysLysHisGluLeuTyrVal    325330335    SerPheArgAspLeuGlyTrpGlnAspTrpIleIleAlaProGluGly    340345350    TyrAlaAlaTyrTyrCysGluGlyGluCysAlaPheProLeuAsnSer    355360365    TyrMetAsnAlaThrAsnHisAlaIleValGlnThrLeuValHisPhe    370375380    IleAsnProAspThrValProLysProCysCysAlaProThrGlnLeu    385390395400    AsnAlaIleSerValLeuTyrPheAspAspSerSerAsnValIleLeu    405410415    LysLysTyrArgAsnMetValValArgAlaCysGlyCysHis    420425430    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1723 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 490..1696    (D) OTHER INFORMATION: /function="OSTEOGENIC PROTEIN"    /product= "hOP2-PP"    /note= "hOP2 cDNA"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    GGCGCCGGCAGAGCAGGAGTGGCTGGAGGAGCTGTGGTTGGAGCAGGAGGTGGCACGGCA60    GGGCTGGAGGGCTCCCTATGAGTGGCGGAGACGGCCCAGGAGGCGCTGGAGCAACAGCTC120    CCACACCGCACCAAGCGGTGGCTGCAGGAGCTCGCCCATCGCCCCTGCGCTGCTCGGACC180    GCGGCCACAGCCGGACTGGCGGGTACGGCGGCGACAGAGGCATTGGCCGAGAGTCCCAGT240    CCGCAGAGTAGCCCCGGCCTCGAGGCGGTGGCGTCCCGGTCCTCTCCGTCCAGGAGCCAG300    GACAGGTGTCGCGCGGCGGGGCTCCAGGGACCGCGCCTGAGGCCGGCTGCCCGCCCGTCC360    CGCCCCGCCCCGCCGCCCGCCGCCCGCCGAGCCCAGCCTCCTTGCCGTCGGGGCGTCCCC420    AGGCCCTGGGTCGGCCGCGGAGCCGATGCGCGCCCGCTGAGCGCCCCAGCTGAGCGCCCC480    CGGCCTGCCATGACCGCGCTCCCCGGCCCGCTCTGGCTCCTGGGCCTG528    MetThrAlaLeuProGlyProLeuTrpLeuLeuGlyLeu    1510    GCGCTATGCGCGCTGGGCGGGGGCGGCCCCGGCCTGCGACCCCCGCCC576    AlaLeuCysAlaLeuGlyGlyGlyGlyProGlyLeuArgProProPro    152025    GGCTGTCCCCAGCGACGTCTGGGCGCGCGCGAGCGCCGGGACGTGCAG624    GlyCysProGlnArgArgLeuGlyAlaArgGluArgArgAspValGln    30354045    CGCGAGATCCTGGCGGTGCTCGGGCTGCCTGGGCGGCCCCGGCCCCGC672    ArgGluIleLeuAlaValLeuGlyLeuProGlyArgProArgProArg    505560    GCGCCACCCGCCGCCTCCCGGCTGCCCGCGTCCGCGCCGCTCTTCATG720    AlaProProAlaAlaSerArgLeuProAlaSerAlaProLeuPheMet    657075    CTGGACCTGTACCACGCCATGGCCGGCGACGACGACGAGGACGGCGCG768    LeuAspLeuTyrHisAlaMetAlaGlyAspAspAspGluAspGlyAla    808590    CCCGCGGAGCGGCGCCTGGGCCGCGCCGACCTGGTCATGAGCTTCGTT816    ProAlaGluArgArgLeuGlyArgAlaAspLeuValMetSerPheVal    95100105    AACATGGTGGAGCGAGACCGTGCCCTGGGCCACCAGGAGCCCCATTGG864    AsnMetValGluArgAspArgAlaLeuGlyHisGlnGluProHisTrp    110115120125    AAGGAGTTCCGCTTTGACCTGACCCAGATCCCGGCTGGGGAGGCGGTC912    LysGluPheArgPheAspLeuThrGlnIleProAlaGlyGluAlaVal    130135140    ACAGCTGCGGAGTTCCGGATTTACAAGGTGCCCAGCATCCACCTGCTC960    ThrAlaAlaGluPheArgIleTyrLysValProSerIleHisLeuLeu    145150155    AACAGGACCCTCCACGTCAGCATGTTCCAGGTGGTCCAGGAGCAGTCC1008    AsnArgThrLeuHisValSerMetPheGlnValValGlnGluGlnSer    160165170    AACAGGGAGTCTGACTTGTTCTTTTTGGATCTTCAGACGCTCCGAGCT1056    AsnArgGluSerAspLeuPhePheLeuAspLeuGlnThrLeuArgAla    175180185    GGAGACGAGGGCTGGCTGGTGCTGGATGTCACAGCAGCCAGTGACTGC1104    GlyAspGluGlyTrpLeuValLeuAspValThrAlaAlaSerAspCys    190195200205    TGGTTGCTGAAGCGTCACAAGGACCTGGGACTCCGCCTCTATGTGGAG1152    TrpLeuLeuLysArgHisLysAspLeuGlyLeuArgLeuTyrValGlu    210215220    ACTGAGGACGGGCACAGCGTGGATCCTGGCCTGGCCGGCCTGCTGGGT1200    ThrGluAspGlyHisSerValAspProGlyLeuAlaGlyLeuLeuGly    225230235    CAACGGGCCCCACGCTCCCAACAGCCTTTCGTGGTCACTTTCTTCAGG1248    GlnArgAlaProArgSerGlnGlnProPheValValThrPhePheArg    240245250    GCCAGTCCGAGTCCCATCCGCACCCCTCGGGCAGTGAGGCCACTGAGG1296    AlaSerProSerProIleArgThrProArgAlaValArgProLeuArg    255260265    AGGAGGCAGCCGAAGAAAAGCAACGAGCTGCCGCAGGCCAACCGACTC1344    ArgArgGlnProLysLysSerAsnGluLeuProGlnAlaAsnArgLeu    270275280285    CCAGGGATCTTTGATGACGTCCACGGCTCCCACGGCCGGCAGGTCTGC1392    ProGlyIlePheAspAspValHisGlySerHisGlyArgGlnValCys    290295300    CGTCGGCACGAGCTCTACGTCAGCTTCCAGGACCTCGGCTGGCTGGAC1440    ArgArgHisGluLeuTyrValSerPheGlnAspLeuGlyTrpLeuAsp    305310315    TGGGTCATCGCTCCCCAAGGCTACTCGGCCTATTACTGTGAGGGGGAG1488    TrpValIleAlaProGlnGlyTyrSerAlaTyrTyrCysGluGlyGlu    320325330    TGCTCCTTCCCACTGGACTCCTGCATGAATGCCACCAACCACGCCATC1536    CysSerPheProLeuAspSerCysMetAsnAlaThrAsnHisAlaIle    335340345    CTGCAGTCCCTGGTGCACCTGATGAAGCCAAACGCAGTCCCCAAGGCG1584    LeuGlnSerLeuValHisLeuMetLysProAsnAlaValProLysAla    350355360365    TGCTGTGCACCCACCAAGCTGAGCGCCACCTCTGTGCTCTACTATGAC1632    CysCysAlaProThrLysLeuSerAlaThrSerValLeuTyrTyrAsp    370375380    AGCAGCAACAACGTCATCCTGCGCAAAGCCCGCAACATGGTGGTCAAG1680    SerSerAsnAsnValIleLeuArgLysAlaArgAsnMetValValLys    385390395    GCCTGCGGCTGCCACTGAGTCAGCCCGCCCAGCCCTACTGCAG1723    AlaCysGlyCysHis    400    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 402 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    MetThrAlaLeuProGlyProLeuTrpLeuLeuGlyLeuAlaLeuCys    151015    AlaLeuGlyGlyGlyGlyProGlyLeuArgProProProGlyCysPro    202530    GlnArgArgLeuGlyAlaArgGluArgArgAspValGlnArgGluIle    354045    LeuAlaValLeuGlyLeuProGlyArgProArgProArgAlaProPro    505560    AlaAlaSerArgLeuProAlaSerAlaProLeuPheMetLeuAspLeu    65707580    TyrHisAlaMetAlaGlyAspAspAspGluAspGlyAlaProAlaGlu    859095    ArgArgLeuGlyArgAlaAspLeuValMetSerPheValAsnMetVal    100105110    GluArgAspArgAlaLeuGlyHisGlnGluProHisTrpLysGluPhe    115120125    ArgPheAspLeuThrGlnIleProAlaGlyGluAlaValThrAlaAla    130135140    GluPheArgIleTyrLysValProSerIleHisLeuLeuAsnArgThr    145150155160    LeuHisValSerMetPheGlnValValGlnGluGlnSerAsnArgGlu    165170175    SerAspLeuPhePheLeuAspLeuGlnThrLeuArgAlaGlyAspGlu    180185190    GlyTrpLeuValLeuAspValThrAlaAlaSerAspCysTrpLeuLeu    195200205    LysArgHisLysAspLeuGlyLeuArgLeuTyrValGluThrGluAsp    210215220    GlyHisSerValAspProGlyLeuAlaGlyLeuLeuGlyGlnArgAla    225230235240    ProArgSerGlnGlnProPheValValThrPhePheArgAlaSerPro    245250255    SerProIleArgThrProArgAlaValArgProLeuArgArgArgGln    260265270    ProLysLysSerAsnGluLeuProGlnAlaAsnArgLeuProGlyIle    275280285    PheAspAspValHisGlySerHisGlyArgGlnValCysArgArgHis    290295300    GluLeuTyrValSerPheGlnAspLeuGlyTrpLeuAspTrpValIle    305310315320    AlaProGlnGlyTyrSerAlaTyrTyrCysGluGlyGluCysSerPhe    325330335    ProLeuAspSerCysMetAsnAlaThrAsnHisAlaIleLeuGlnSer    340345350    LeuValHisLeuMetLysProAsnAlaValProLysAlaCysCysAla    355360365    ProThrLysLeuSerAlaThrSerValLeuTyrTyrAspSerSerAsn    370375380    AsnValIleLeuArgLysAlaArgAsnMetValValLysAlaCysGly    385390395400    CysHis    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1926 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 93..1289    (D) OTHER INFORMATION: /function="OSTEOGENIC PROTEIN"    /product= "mOP2-PP"    /note= "mOP2 cDNA"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    GCCAGGCACAGGTGCGCCGTCTGGTCCTCCCCGTCTGGCGTCAGCCGAGCCCGACCAGCT60    ACCAGTGGATGCGCGCCGGCTGAAAGTCCGAGATGGCTATGCGTCCCGGGCCA113    MetAlaMetArgProGlyPro    15    CTCTGGCTATTGGGCCTTGCTCTGTGCGCGCTGGGAGGCGGCCACGGT161    LeuTrpLeuLeuGlyLeuAlaLeuCysAlaLeuGlyGlyGlyHisGly    101520    CCGCGTCCCCCGCACACCTGTCCCCAGCGTCGCCTGGGAGCGCGCGAG209    ProArgProProHisThrCysProGlnArgArgLeuGlyAlaArgGlu    253035    CGCCGCGACATGCAGCGTGAAATCCTGGCGGTGCTCGGGCTACCGGGA257    ArgArgAspMetGlnArgGluIleLeuAlaValLeuGlyLeuProGly    40455055    CGGCCCCGACCCCGTGCACAACCCGCCGCTGCCCGGCAGCCAGCGTCC305    ArgProArgProArgAlaGlnProAlaAlaAlaArgGlnProAlaSer    606570    GCGCCCCTCTTCATGTTGGACCTATACCACGCCATGACCGATGACGAC353    AlaProLeuPheMetLeuAspLeuTyrHisAlaMetThrAspAspAsp    758085    GACGGCGGGCCACCACAGGCTCACTTAGGCCGTGCCGACCTGGTCATG401    AspGlyGlyProProGlnAlaHisLeuGlyArgAlaAspLeuValMet    9095100    AGCTTCGTCAACATGGTGGAACGCGACCGTACCCTGGGCTACCAGGAG449    SerPheValAsnMetValGluArgAspArgThrLeuGlyTyrGlnGlu    105110115    CCACACTGGAAGGAATTCCACTTTGACCTAACCCAGATCCCTGCTGGG497    ProHisTrpLysGluPheHisPheAspLeuThrGlnIleProAlaGly    120125130135    GAGGCTGTCACAGCTGCTGAGTTCCGGATCTACAAAGAACCCAGCACC545    GluAlaValThrAlaAlaGluPheArgIleTyrLysGluProSerThr    140145150    CACCCGCTCAACACAACCCTCCACATCAGCATGTTCGAAGTGGTCCAA593    HisProLeuAsnThrThrLeuHisIleSerMetPheGluValValGln    155160165    GAGCACTCCAACAGGGAGTCTGACTTGTTCTTTTTGGATCTTCAGACG641    GluHisSerAsnArgGluSerAspLeuPhePheLeuAspLeuGlnThr    170175180    CTCCGATCTGGGGACGAGGGCTGGCTGGTGCTGGACATCACAGCAGCC689    LeuArgSerGlyAspGluGlyTrpLeuValLeuAspIleThrAlaAla    185190195    AGTGACCGATGGCTGCTGAACCATCACAAGGACCTGGGACTCCGCCTC737    SerAspArgTrpLeuLeuAsnHisHisLysAspLeuGlyLeuArgLeu    200205210215    TATGTGGAAACCGCGGATGGGCACAGCATGGATCCTGGCCTGGCTGGT785    TyrValGluThrAlaAspGlyHisSerMetAspProGlyLeuAlaGly    220225230    CTGCTTGGACGACAAGCACCACGCTCCAGACAGCCTTTCATGGTAACC833    LeuLeuGlyArgGlnAlaProArgSerArgGlnProPheMetValThr    235240245    TTCTTCAGGGCCAGCCAGAGTCCTGTGCGGGCCCCTCGGGCAGCGAGA881    PhePheArgAlaSerGlnSerProValArgAlaProArgAlaAlaArg    250255260    CCACTGAAGAGGAGGCAGCCAAAGAAAACGAACGAGCTTCCGCACCCC929    ProLeuLysArgArgGlnProLysLysThrAsnGluLeuProHisPro    265270275    AACAAACTCCCAGGGATCTTTGATGATGGCCACGGTTCCCGCGGCAGA977    AsnLysLeuProGlyIlePheAspAspGlyHisGlySerArgGlyArg    280285290295    GAGGTTTGCCGCAGGCATGAGCTCTACGTCAGCTTCCGTGACCTTGGC1025    GluValCysArgArgHisGluLeuTyrValSerPheArgAspLeuGly    300305310    TGGCTGGACTGGGTCATCGCCCCCCAGGGCTACTCTGCCTATTACTGT1073    TrpLeuAspTrpValIleAlaProGlnGlyTyrSerAlaTyrTyrCys    315320325    GAGGGGGAGTGTGCTTTCCCACTGGACTCCTGTATGAACGCCACCAAC1121    GluGlyGluCysAlaPheProLeuAspSerCysMetAsnAlaThrAsn    330335340    CATGCCATCTTGCAGTCTCTGGTGCACCTGATGAAGCCAGATGTTGTC1169    HisAlaIleLeuGlnSerLeuValHisLeuMetLysProAspValVal    345350355    CCCAAGGCATGCTGTGCACCCACCAAACTGAGTGCCACCTCTGTGCTG1217    ProLysAlaCysCysAlaProThrLysLeuSerAlaThrSerValLeu    360365370375    TACTATGACAGCAGCAACAATGTCATCCTGCGTAAACACCGTAACATG1265    TyrTyrAspSerSerAsnAsnValIleLeuArgLysHisArgAsnMet    380385390    GTGGTCAAGGCCTGTGGCTGCCACTGAGGCCCCGCCCAGCATCCTGCTTCTACT1319    ValValLysAlaCysGlyCysHis    395    ACCTTACCATCTGGCCGGGCCCCTCTCCAGAGGCAGAAACCCTTCTATGTTATCATAGCT1379    CAGACAGGGGCAATGGGAGGCCCTTCACTTCCCCTGGCCACTTCCTGCTAAAATTCTGGT1439    CTTTCCCAGTTCCTCTGTCCTTCATGGGGTTTCGGGGCTATCACCCCGCCCTCTCCATCC1499    TCCTACCCCAAGCATAGACTGAATGCACACAGCATCCCAGAGCTATGCTAACTGAGAGGT1559    CTGGGGTCAGCACTGAAGGCCCACATGAGGAAGACTGATCCTTGGCCATCCTCAGCCCAC1619    AATGGCAAATTCTGGATGGTCTAAGAAGGCCCTGGAATTCTAAACTAGATGATCTGGGCT1679    CTCTGCACCATTCATTGTGGCAGTTGGGACATTTTTAGGTATAACAGACACATACACTTA1739    GATCAATGCATCGCTGTACTCCTTGAAATCAGAGCTAGCTTGTTAGAAAAAGAATCAGAG1799    CCAGGTATAGCGGTGCATGTCATTAATCCCAGCGCTAAAGAGACAGAGACAGGAGAATCT1859    CTGTGAGTTCAAGGCCACATAGAAAGAGCCTGTCTCGGGAGCAGGAAAAAAAAAAAAAAC1919    GGAATTC1926    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 399 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    MetAlaMetArgProGlyProLeuTrpLeuLeuGlyLeuAlaLeuCys    151015    AlaLeuGlyGlyGlyHisGlyProArgProProHisThrCysProGln    202530    ArgArgLeuGlyAlaArgGluArgArgAspMetGlnArgGluIleLeu    354045    AlaValLeuGlyLeuProGlyArgProArgProArgAlaGlnProAla    505560    AlaAlaArgGlnProAlaSerAlaProLeuPheMetLeuAspLeuTyr    65707580    HisAlaMetThrAspAspAspAspGlyGlyProProGlnAlaHisLeu    859095    GlyArgAlaAspLeuValMetSerPheValAsnMetValGluArgAsp    100105110    ArgThrLeuGlyTyrGlnGluProHisTrpLysGluPheHisPheAsp    115120125    LeuThrGlnIleProAlaGlyGluAlaValThrAlaAlaGluPheArg    130135140    IleTyrLysGluProSerThrHisProLeuAsnThrThrLeuHisIle    145150155160    SerMetPheGluValValGlnGluHisSerAsnArgGluSerAspLeu    165170175    PhePheLeuAspLeuGlnThrLeuArgSerGlyAspGluGlyTrpLeu    180185190    ValLeuAspIleThrAlaAlaSerAspArgTrpLeuLeuAsnHisHis    195200205    LysAspLeuGlyLeuArgLeuTyrValGluThrAlaAspGlyHisSer    210215220    MetAspProGlyLeuAlaGlyLeuLeuGlyArgGlnAlaProArgSer    225230235240    ArgGlnProPheMetValThrPhePheArgAlaSerGlnSerProVal    245250255    ArgAlaProArgAlaAlaArgProLeuLysArgArgGlnProLysLys    260265270    ThrAsnGluLeuProHisProAsnLysLeuProGlyIlePheAspAsp    275280285    GlyHisGlySerArgGlyArgGluValCysArgArgHisGluLeuTyr    290295300    ValSerPheArgAspLeuGlyTrpLeuAspTrpValIleAlaProGln    305310315320    GlyTyrSerAlaTyrTyrCysGluGlyGluCysAlaPheProLeuAsp    325330335    SerCysMetAsnAlaThrAsnHisAlaIleLeuGlnSerLeuValHis    340345350    LeuMetLysProAspValValProLysAlaCysCysAlaProThrLys    355360365    LeuSerAlaThrSerValLeuTyrTyrAspSerSerAsnAsnValIle    370375380    LeuArgLysHisArgAsnMetValValLysAlaCysGlyCysHis    385390395    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 6418 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1..6361    (D) OTHER INFORMATION: /note= "hOP-2 genomic sequence"    (ix) FEATURE:    (A) NAME/KEY: exon    (B) LOCATION: 1..837    (D) OTHER INFORMATION: /note= "EXON ONE"    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 884..885    (D) OTHER INFORMATION: /note= "A GAP OCCURS BETWEEN    POSTIONS 884 AND 885 IN THIS SEQUENCE"    (ix) FEATURE:    (A) NAME/KEY: exon    (B) LOCATION: 1088..1277    (D) OTHER INFORMATION: /note= "EXON TWO"    (ix) FEATURE:    (A) NAME/KEY: exon    (B) LOCATION: 1350..1814    (D) OTHER INFORMATION: /note= "EXON THREE"    (ix) FEATURE:    (A) NAME/KEY: misc.sub.-- feature    (B) LOCATION: 1834..1835    (D) OTHER INFORMATION: /note= "A GAP OCCURS BETWEEN    POSITIONS 1834 AND 1835 IN THIS SEQUENCE"    (ix) FEATURE:    (A) NAME/KEY: exon    (B) LOCATION: 1883..2077    (D) OTHER INFORMATION: /note= "EXON FOUR"    (ix) FEATURE:    (A) NAME/KEY: exon    (B) LOCATION: 2902..2981    (D) OTHER INFORMATION: /note= "EXON FIVE"    (ix) FEATURE:    (A) NAME/KEY: exon    (B) LOCATION: 3507..3617    (D) OTHER INFORMATION: /note= "EXON SIX"    (ix) FEATURE:    (A) NAME/KEY: exon    (B) LOCATION: 6116..6361    (D) OTHER INFORMATION: /note= "EXON SEVEN"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    GGAATTCCGGCCACAGTGGCGCCGGCAGAGCAGGAGTGGCTGGAGGAGCTGTGGTTGGAG60    CAGGAGGTGGCACGGCAGGGCTGGAGGGCTCCCTATGAGTGGCGGAGACGGCCCAGGAGG120    CGCTGGAGCAACAGCTCCCACACCGCACCAAGCGGTGGCTGCAGGAGCTCGCCCATCGCC180    CCTGCGCTGCTCGGACCGCGGCCACAGCCGGACTGGCGGGTACGGCGGCGACAGACGGAT240    TGGCCGAGAGTCCCAGTCCGCAGAGTAGCCCCGGCCTCGAGGCGGTGGCGTCCGCGTCCT300    CTCGTCCAGGAGCCAGGACAGGTGTCGCGCGGCGGGCCGTCCAGGGACCGCGCTGAGGCC360    GCGGTCGCCCGTCCCGCCCCGCCCCGCCGCCCGCCGCCCGCCGAGCCCAGCCTCCTTGCC420    GTCGGGGCGTCCCCAGGCCCTGGGTCGGCCGCGGAGCCGATGCGCGCCCGCTGAGCGCCC480    CAGCTGAGCGCCCCCGGCCTGCCATGACCGCGCTCCCCGGCCCGCTCTGGCTCCTGGGCC540    TGGCGCTATGCGCGCTGGGCGGGGGCGGCCCCGGCCTGCGACCCCCGCCCGGCTGTCCCC600    AGCGACGTCTGGGCGCGCGCGAGCGCCGGGACGTGCAGCGCGAGATCCTGGCGGTGCTCG660    GGCTGCCTGGGCGGCCCCGGCCCCGCGCGCCACCCGCCGCCTCCCGGCTGCCCGCGTCCG720    CGCCGCTCTTCATGCTGGACCTGTACCACGCCATGGCCGGCGACGACGACGAGGACGGCG780    CGCCCGCGGAGCGGCGCCTGGGCCGCGCCGACCTGGTCATGAGCTTCGTTAACATGGGTG840    AGTGCGGCGCCCGCGCGGGGACCCTCGGAGTAAACTGGCTGCAGCTGCAGGGCCTCTTCT900    GGCTCTACACCCCGGGACCAAGCCTGGAACAAACGTTTGCACTAAATGAAGCCGGCCCCA960    CCCAGGCCTCCCTGGGTCCGCTCCACCTTGAGTGGTGGGTGGCTGGGGGCGGTGGCTCAC1020    ACCAGCTCTGCCCCCTCCAGAGCCCGAGCCATTCTGAGTGCCAGCCCAGCGCTGCTTTGT1080    CTTCTAGTGGAGCGAGACCGTGCCCTGGGCCACCAGGAGCCCCATTGGAAGGAGTTCCGC1140    TTTGACCTGACCCAGATCCCGGCTGGGGAGGCGGTCACAGCTGCGGAGTTCCGGATTTAC1200    AAGGTGCCCAGCATCCACCTGCTCAACAGGACCCTCCACGTCAGCATGTTCCAGGTGGTC1260    CAGGAGCAGTCCAACAGGTGCCTTCCCCTTGGCCCGGGTGCCCACCTAACCCCCCACCTC1320    ACAGTCTCATGGTCAAGGCAGCCCAGCAGGGAGTCGTGGTGGGTGAAAGAGAGCCTCAAA1380    GATGGGAAGGATGCTTGGCCCGAGGCCCTGCACTGTGGGAAGAGCCCCAGTGACAATCCT1440    GACTTCAAGTCCCTGCCCTCCATCCTGCTGTGGGGACTTGGACATGGTCACTGAGACTCA1500    GTTTCCCCATGTGTACACCTCTGTGGGCTGAGGCAATGAGATGAGGCTCAGAAGGGCGCA1560    GCCAGAGTCAGGTGGGAGACGCTCCGGTGACAGCCCCCAGCGGGCCCTGGAGACACGGAG1620    GCAGCTGTGCCGGCCGCCGGTTAATTGTTCTTTCATGTCCACAGGGGAGTCTGACTTGTT1680    CTTTTTGGATCTTCAGACGCTCCGAGCTGGAGACGAGGGCTGGCTGGTGCTGGATGTCAC1740    AGCAGCCAGTGACTGCTGGTTGCTGAAGCGTCACAAGGACCTGGGACTCCGCCTCTATGT1800    GGAGACTGAGGACGGTGAGGCTGGGGCTCTGCAGCTGCAGAGCCACTGCCCGTGAGTGAC1860    CCCTCTCTCCTTTCTGTCTCAGGGCACAGCGTGGATCCTGGCCTGGCCGGCCTGCTGGGT1920    CAACGGGCCCCACGCTCCCAACAGCCTTTCGTGGTCACTTTCTTCAGGGCCAGTCCGAGT1980    CCCATCCGCACCCCTCGGGCAGTGAGGCCACTGAGGAGGAGGCAGCCGAAGAAAAGCAAC2040    GAGCTGCCGCAGGCCAACCGACTCCCAGGGATCTTTGGTGAGGGTCGGGCAGGCTGGGGC2100    GAGGCTGTGGCTGTCTGGCTGAGAGAGGCAGGGCGAGAACCAAGTGGTGGCCCAGAGCCC2160    AGAGCCTCAGGCTAGGTCGGTTCAAGCTGACGGCCACTCTCCAGCCACCTTTCCTGACAC2220    CATCTTGGCCCTGATGCACCCTGGTGACCGGCACTCCGAGGCCTGTCCTGGCTGTCCCTG2280    CTGCCAGAAGTCTCCCTCTCTCCCCCTGGCTCCTCCGGGTCTTTCTCAGGAGCCTCCTTC2340    AGAATCAGCTGCCCCTTCCCTGGGAGCCGCAGCCCCTCATGACCTGCGGTTGTGCCTGGG2400    CACCTGTGGATCCTCGGTTGCTTATGCGATTTTCTCCCCAACTGGCCAAGCTTCAGGATC2460    AGGGACAGGCCTGACCCAACCCCGTGCCCTCCTTCCCAGGGAGTCGGCCCTTGACTGGCC2520    TGGTCGTGAGCCACTTGAACCTCGGGAATGGGTGTGGCAGGAGAGGGTGGGCTGGAGTCA2580    CAGGGGTCTCCAGAGAGGAGGAGGCACAGGATGGCCGAGGGTCCTGCTGGGCTGTTTACT2640    GGAGCATAAAGATGCTCATAGGCTGAAGGACAGGGGAGGACTGGGCACAGTGTCACTCTA2700    GCCATTGGGAGCCATGGCAGGCTTCTGAGCTGGGTCATGGTACAAGCAGAGTTCCAGGGA2760    TGGGCTTTATGAGCCAAATGGTTTCCTGTCATTCATTTATTTGACAAATGTGCTCATCAG2820    GGCATCCCCCACCCTGGTACCCCATAGTAGCTGCACACAGCAGGAACCCCAGAAAAGACC2880    TTGCCCCTTCTGTCCCTGCAGATGACGTCCACGGCTCCCACGGCCGGCAGGTCTGCCGTC2940    GGCACGAGCTCTACGTCAGCTTCCAGGACCTCGGCTGGCTGGTAATTGCTGACTCTCCTT3000    GTTTCTGAAATGACAATCACCACCTGTAGATCAGAAGTGAATCTGCAGGGAGGACATAGA3060    ATCATGGTGACTTCAATTTTCTTATGTATTTTTTTCTTCTGTGTTTTCCAAGTTTTCTAA3120    AGTGAGAATATGGTGAGAAAGGGTTTTGTTGTTGTTGTTGTGTTTTTTGTTTTTTTTTAA3180    AAACCCATGAAAATGAAGACTGAATCAACCAACTAAGCTGTCAGCATTGCCGCAGGGTAA3240    CTGAGACCTCCCTGCATTGGCTACGACTGCAGCTCTGGGAGGTGTGGGCAGGGGAGGGCC3300    GGCTGGGGAGGGCCGGCTGGGGAGGGGACACAAAGTGAAGATGGGGGTTGTTGGGCCTGA3360    GCTCCTGCCCAGCCTTTTCCGCCGGGGTTCCTGGGTGGATTCAAGCCTCTTGGGGGAGAC3420    GCGCTGCAGGGCTGGAGGATGGGCTTTGGGCCCTGAGGCTCAGGGAGGAGCACATGGATG3480    GGACTCACCTTCTCCCTTGCCCCCAGGACTGGGTCATCGCTCCCCAAGGCTACTCGGCCT3540    ATTACTGTGAGGGGGAGTGCTCCTTCCCACTGGACTCCTGCATGAATGCCACCAACCACG3600    CCATCCTGCAGTCCCTGGTCGATACCGTCGCCCATCCTGCCCAGCCCCCTGGTGGAGGCC3660    CTGCAGAGAGGGGTCTGGTCCAGCCAGCCGGGAGGCAGTGAGGCCACCTGCTCCATGTCT3720    CGGGGCTTTGTCTGCACAGAGTCAGTAACGTCGCTAACTTCCCACAGCTCTGCAGGAACT3780    GGTCCTCATACAGCCACACTACTACACATAGACCCACACCCAAACACGGACACACGTGAA3840    CAGTCGCGTATCATGCCTGTTCTATGCACTGAACAAACTCCTGTGGGACACTTACACACC3900    TGCGTGCGGCGCTCAGAGGCACAGCACATGAAACAGATGTGTACACTGTGTGGGGGCTGT3960    GTGATCTTAACACACGGGCCCCCGAGTACGCTGGCAAGTCTGACCGCCCGTGATATGTGC4020    GCACAGTGTGTGGGGTGTGCGTGTGCATCACCCACCTGTGCCGCACCACAGGTAGGAAGC4080    TTCTAGATGGTGTGGCTCTCAACCTTTTGGCTTTTTCCCGCAGTTTCTCTCTTGGCTGTC4140    TGTGTTTTCTCTGGATCCCCTGGCTTTTGATGCCGTTGGTGTCTGGGGCAACCTTAAAGG4200    ACAAAAGCAGGCTTCTGATGGGATCACTGGTGCTGCTCACCACTGAGTGCTCGTGTGTTT4260    GCGGATTCTGGCACCGAGGCTTCCTTCTAGAAGTTTTTACCTAGAATCCCAGTTCCTGGT4320    ATTGCACAGCCTTATGTTTTCCTCTTAGGAGGTTCAACGGTGATGCCTTGATCAGGCGCA4380    GTGGCTCACCCTGTAATCGCAGCACACGAGCCCAGAAGTTCAAGACAAGCCTGAGCAACA4440    CAGCAAAACCCTGTCTCTAAAATAAAAATTAAAACACACACACACACACACACACACACA4500    CACACACGTGCGCACACAATGCCTTGGTGTGAGAGGAAAGAAATTACCAAAAGCTGCTCT4560    GAGCCTATGATAATACTTCCTTTCTGGGCAGTCAAATGGTGTTTGCTGGACACCCTGGAG4620    CCATCTCCTTGGAAAGGCCCAGGGGTGATGAGGAGCTCCGTCGGGGTGGCCTGGCCAGCA4680    CCTTTATGCCGTGTGGTTCTCACAGCTGCATGTGTGGGAGGTACATGGGAAGGTGACTGC4740    ACCTGCGCTCCTGGACTCCATCTCCTCTGCCCTTGCCCCTGCCCCTCACGTGCAACTAGA4800    GTGAGTGCTCACAGCCTACAGGGCAGCAAACAGGCACTGTGCTCTAGGGGAGGCTGTCGG4860    TGGGCACAGAAGCAAACCAACCGTGGAGTTGACACCTCCTGTGAGGAAGAGCAGACGAGC4920    CGTGCCGTCAGTGGAGTGAGACTGGGCCCAGCTCTCCACACAAGGAGGGGCACGTCAGCA4980    GCTGGAGGAGGAATGTTCCAGAAGGAGCAAGTGCAAGGCCCTAAGACAGGAGCAGGCTGG5040    CCCTAAGTTCAGGGCAGGGGAGGAGAGGGGCTGGGTGCAGTGAAGGGGAGGAGAGTGGAG5100    GGAGGTGATCCGGGGTGATAGGCCAGCTCCCGTAGCCTGGGTTCCCTGGGAAGAGGGTGG5160    ATTTTATTCCAAGCAACCCCAGAGGCTGTCAGAGGTCTTCAGCAAAGAGTGTCCTTGGTC5220    TGCGTCACCCTCCAGAAGGACCTTTCTGGCTTGGGGAGGTCGCGGGAGTGGAAGGCAGAG5280    GAGCAGGGGATGAGTGAGGGCTGCTGTGGTCACCTGGCAGGTGATGGCAGCTCGACTGGG5340    CAGGTGGTCCGAGGCAGCACGGAGGTGGAGGTTGAGCCAGGGGCTGCTCTCAGGGAAGGG5400    AGGAGGCGAAAGGAGTCATCCAGGAGGCCTCCCAGGCGGGAGCTATGATGTCAGGGCGGG5460    AGGAATTCTATGTTCCACTGAGGCCTCATTAGACCCCCAAGTGCAGAAGTGGGAAGGGGA5520    GCAGGATCCGCAAGTCTGGAGTTCAGAAGAGAGGTCCAAGCTGAGCCAGGGGAGTGGAGA5580    GGTGCGGGCCAATGCAGGGCCTTGAAGTGCTGAGGGCGGATCGAGTCCTCTGGGAGAAGG5640    AGCAGCACAGGAGAGGGGGCGAGGCTGGCTCCCAGAGCCTGGGGAGGGAGGCAGGTGTGG5700    GGAGGCAGAGCTTGGGGGGGTCTGAAGGGCTATAAGAAGACAGTGGTCCTTCCAGGTTCC5760    CCCTTGGACCTCACTAAGGGCACAAACCTGGCCATGAGGTTCTCCTTCCCATTATCCCCA5820    GGAGGAAGTCTGAGCCCTTGGCCTGGGACTCGAGGCCCCTCATTAGTGCCCTGCCCACCT5880    GCCCCACACCCTGGGGCTGCCATGTATCCCTCCCTGGGCACTGTGGGCACCACAGCTCCC5940    GCTCCCAGAGCTCTCAGGGCTGCTCTTATTCCTGTTAATAATTCTTATTATTGTGCTGCT6000    CCCATGTGGCTTGGAGATGGCCAGGGCAGGGAGCAGGTGGAGCTGGGGCGGGCTAGGTGG6060    GTCCTCAGAGGAGGCCACTGGCTCATGCCCCTGCCTGTGCTCCCTTCCTGGCCAGGTGCA6120    CCTGATGAAGCCAAACGCAGTCCCCAAGGCGTGCTGTGCACCCACCAAGCTGAGCGCCAC6180    CTCTGTGCTCTACTATGACAGCAGCAACAACGTCATCCTGCGCAAGCACCGCAACATGGT6240    GGTCAAGGCCTGCGGCTGCCACTGAGTCAGCCCGCCCAGCCCTACTGCAGCCACCCTTCT6300    CATCTGGATCGGGCCCTGCAGAGGCAGAAAACCCTTAAATGCTGTCACAGCTCAAGCAGG6360    AGTGTCAGGGGCCCTCACTCTCTGTGCCTACTTCCTGTCAGGCTTCTGGTCCTTTCTC6418    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 97 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (ix) FEATURE:    (A) NAME/KEY: Protein    (B) LOCATION: 1..97    (D) OTHER INFORMATION: /label=Generic-Seq-7    /note= "wherein each Xaa is independently selected from    a group of one or more specified amino acids as defined    in the specification."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    LeuXaaXaaXaaPheXaaXaaXaaGlyTrpXaaXaaXaaXaaXaaXaa    151015    ProXaaXaaXaaXaaAlaXaaTyrCysXaaGlyXaaCysXaaXaaPro    202530    XaaXaaXaaXaaXaaXaaXaaXaaAsnHisAlaXaaXaaXaaXaaXaa    354045    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaCysCysXaaPro    505560    XaaXaaXaaXaaXaaXaaXaaXaaLeuXaaXaaXaaXaaXaaXaaXaa    65707580    ValXaaLeuXaaXaaXaaXaaXaaMetXaaValXaaXaaCysXaaCys    859095    Xaa    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 102 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (ix) FEATURE:    (A) NAME/KEY: Protein    (B) LOCATION: 1..102    (D) OTHER INFORMATION: /label=Generic-Seq-8    /note= "wherein each Xaa is independently selected from    a group of one or more specified amino acids as defined    in the specification."    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    CysXaaXaaXaaXaaLeuXaaXaaXaaPheXaaXaaXaaGlyTrpXaa    151015    XaaXaaXaaXaaXaaProXaaXaaXaaXaaAlaXaaTyrCysXaaGly    202530    XaaCysXaaXaaProXaaXaaXaaXaaXaaXaaXaaXaaAsnHisAla    354045    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa    505560    XaaCysCysXaaProXaaXaaXaaXaaXaaXaaXaaXaaLeuXaaXaa    65707580    XaaXaaXaaXaaXaaValXaaLeuXaaXaaXaaXaaXaaMetXaaVal    859095    XaaXaaCysXaaCysXaa    100    __________________________________________________________________________

What is claimed is:
 1. An isolated protein, the amino acid sequence ofwhich comprises the sequence shown in residues 303 to 399 of Seq. ID No.1, or a conservative variant thereof.
 2. The protein of claim 1 whereinsaid amino acid sequence comprises the sequence shown in residues 298 to399 of Seq. ID No. 1, or a conservative variant thereof.
 3. The proteinof claim 2 wherein said amino acid sequence comprises the sequence shownin residues 264 to 399 of Seq. ID No. 1, or a conservative variantthereof.
 4. The protein of claim 3 wherein said amino acid sequencecomprises the sequence shown in residues 261 to 399 of Seq. ID No. 1, ora conservative variant thereof.
 5. The protein of claim 4 wherein saidamino acid sequence comprises the sequence shown in residues 18 to 399of Seq. ID No. 1, or a conservative variant thereof.
 6. The protein ofclaim 5 wherein said amino acid sequence comprises the sequence shown inresidues 1 to 399 of Seq. ID No. 1, or a conservative variant thereof.7. A chimeric morphogen comprising a polypeptide having the amino acidsequence of claim 1, 2 or
 3. 8. The protein of claim 1, 2 or 3 whereinsaid conservative variant has an amino acid substitution for the serineat position 315 or the cysteine at position 338 in Seq. ID No.
 1. 9. Theprotein of claim 8 wherein said conservative variant has a tryptophanresidue in place of the serine at position 315 in Seq. ID No.
 1. 10. Theprotein of claim 8 wherein said cysteine residue at position 338 in Seq.ID No. 1 is substituted by an amino acid selected from the groupconsisting of tyrosine, histidine, isoleucine and serine.
 11. Anisolated dimeric protein comprising a pair of polypeptides encoded bynucleic acid, the sequence of which comprises at least the nucleic acidsequence of bases 960-1265 of Seq. ID No. 1, or a conservative variantthereof, such that said dimeric protein induces tissue-specificmorphogenesis of mammalian tissue.
 12. An isolated dimeric proteincomprising a first polypeptide, the amino acid sequence of whichcomprises the sequence shown in residues 303 to 399 of Seq. ID No. 1 ora conservative variant thereof, and a second polypeptide the amino acidsequence of which comprises the sequence shown in Generic Sequence 7 or8 (Seq. ID Nos. 12 or 13) such that said dimeric protein inducestissue-specific morphogenesis of mammalian tissue.